Towards sarcosine determination in urine for prostatic carcinoma detection

Sarcosine, a potential biomarker for prostate cancer, can be detected in a solid state enzyme based biosensor using sarcosine oxidase, with particle immobilised reagents. A novel fusion protein of the fluorescent protein, mCherry, sarcosine oxidase (SOx), and the polypeptide R5 (R52-mCherry-SOx-R5-6H), was explored, which allowed self-immobilization on silica microparticles and long-term (90 days +) retention of activity, even at room temperature. In contrast, commercial wildtype SOx lost activity in a few days. A silica-R52-mCherry-SOx-R5-6H microparticle sensor for determination of sarcosine in urine, linked the SOx coproduct, H2O2, to a measurement catalysed by horseradish peroxidase (HRP) immobilised on silica, in the presence of Amplex Ultrared (AR) to generate fluorescence at 582 nm. Silica microparticles carrying all the reagents (R52-mCherry-SOx-R5-6H, HRP and AR) were used to produce a silica-microparticle biosensor which responded to sarcosine at micromolar levels. Interference by amino acids and uric acid was examined and it was found that the silica-reagent carrying system could be calibrated in urine and responded across the clinically relevant concentration range. This contrasted with similar assays using commercial SOx, where interference inhibited the sarcosine signal measurement in urine. The microparticle biosensor was tested in urine from healthy volunteers and prostate cancer patients, showing higher concentrations of sarcosine in cancer patients consistent with previous reports of elevated sarcosine levels.BBSRC/EPSRC funded Grant No. BB/L014130/1 Gates Cambridge Trust Generalitat Valenciana and European commission for its postdoctoral grant (APOSTD/113/2016) Generalitat Valenciana (PROMETEO 2016/109

Ionic-liquid doped polymeric composite as passive colorimetric sensor for meat freshness as a use case

[EN] A composite membrane containing 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS) embedded in an ionic liquid (IL)- polydimethylsiloxane (PDMS)- tetraethyl orthosilicate (TEOS)- SiO2 nanoparticles (NPs) polymeric matrix is proposed. The selected IL was 1-methyl-3-octylimidazolium hexafluorophosphate (OMIM PF6). It is demonstrated that ILs chemical additives of PDMS influenced the sol-gel porosity. The sensor analytical performance for ammonia atmospheres has been tested as a function of sampling time (between 0.5 and 312 h), temperature (25 ◦C and 4 ◦C) and sampling volume (between 2L and 22 mL) by means of diffuse reflectance measurements and sensor photos, which can be registered and saved as images by a smartphone, which permit RGB measurements too. Flexible calibration was possible, adapting it to the sampling time, temperature and sampling volume needed for its application. Calibration linear slopes (mA vs ppmv) between 1.7 and 467 ppmv-1 were obtained for ammonia in function of the several studied conditions. Those slopes were between 48 and 91% higher than those achieved with sensors without ILs. The practical application of this sensing device was demonstrated for the analysis of meat packaging environments, being a potential cost-effective candidate for in situ meat freshness analysis. NQS provided selectivity in reference to other family compounds emitted from meat products, such as sulphides. After 10 days at 4 ◦C ammonia liberated by the assayed meat was 20 ± 4 μg/kg and 18 ± 3 μg/kg, quantified by using diffuse reflectance and %R measurements, respectively. Homogeneity of the ammonia atmosphere was tested by using two sensors placed in two different positions inside the packages

Zein as biodegradable material for effective delivery of alkaline phosphatase and substrates in biokits and biosensors

A biodegradable material, zein, is proposed as a reagent delivery platform for biokits and biosensors based on alkaline phosphatase (ALP) activity/inhibition in the presence of phosphatase substrates. The immobilization and release of both the substrate and/or the active ALP, in a biodegradable and low-cost material such as zein, a prolamin from maize, and in combination with glycerol as plasticizer have been investigated. Three zein-based devices are proposed for several applications: (1) inorganic phosphorus estimation in water of different sources (river, lake, coastal water and tap water) with a detection limit of 0.2mg/L - compared to at least 1mg/L required by legislation, (2) estimation of ALP in saliva and (3) chlorpyrifos control in commercial preparations. The single-use kits developed are low cost, easy and fast to manufacture and are stable for at least 20 days at -20°C, so the zein film can preserve and deliver both the enzyme and substrates.The authors are grateful to Generalitat Valenciana (PROMETEO 2012/045 project and BEFPI/2014/014), Spain and to the Spanish Ministerio de Economía y Competitividad/FEDER (Project CTQ2014-53916-P). NJ expresses her gratitude to PROMETEO program for her predoctoral grant and BEFPI program for her stay grant

Towards in field miniaturized liquid chromatography: Biocides in wastewater as a proof of concept

[EN] Solving and/or evaluating given problems or decision making in place and in real time is a goal of the an- alytical chemistry science. In this context, the performance of a commercial portable miniaturized liquid chromatograph (minLC) with LED UV (255 nm) detector was compared with those provided by two lab minLCs (capillary and nano) coupled on-line to in-valve in-tube solid phase microextraction (IT-SPME) with diode array detector (DAD). In addition, responses of the portable LC for in-field analysis in sev- eral conditions were tested. Besides, two evaluation tools, BETTER criteria for portability and HEXAGON pictogram for sustainability and greenness were applied for comparison purposes. The benchtop LCs pro- vided lower limits of detection (LODs) as expected, in the order of low μg/L, than those achieved by the portable LC, with LODs around mg/L for compounds covering several polarities (logKow between -1.72 and 3.82). The used portable LC gave excellent resolution, reducing the analysis time and being the consumption of solvents negligible. As a practical application, fruit washing residual waters, which contained a suitable level of concentrations of several biocides for employing the portable minLC, were analyzed and quantified from the three minLCs as a proof of concept with comparable results

Evaluation of the sample treatment influence in green and sustainable assessment of liquid chromatography methods by the HEXAGON tool: Sulfonate-based dyes determination in meat samples

Green and Sustainable assessment of sample treatment has been carried out when using liquid chromatographic methods in food analysis. Particularly, published sulfonate-based dyes determination by conventional liquid chromatography with diode array (HPLC-DAD) or mass detectors (HPLC-MS/MS) was selected as starting point. Greenness-sustainability have been quantitatively evaluated by means of the HEXAGON tool, in which sample treatment and method characteristics noticeably play a key role in defining the figures of merit of the analytical procedure. According to the evaluation results that are represented in a hexagon pictogram with an overall score from 0 to 4 (the lower the score, the greater contribution), a new analytical method is proposed by means of both minimizing and miniaturizing sample treatment step. In-tube solid-phase microextraction online coupled to capillary LC (IT-SPME-CapLC-DAD) provided a more green and sustainable alternative to current food dyes analysis without losing sensitivity (LODs 0.5-1 µg/g sample). The proposed method was tested on chicken meat samples (2 g), using ethanol-ammonia-water mixture (80:1:19, v/v/v; 5 mL) as extraction solution. The arithmetic means of the HEXAGON scores (sav) were: 1.71, 2.57 and 2.71 for IT-SPME-CapLC-DAD, HPLC-DAD and HPLC-MS/MS, respectively. The proposed method was closer than the others tested to a hypothetical LC procedure without sample treatment, which provided a sav value of 1

Towards sarcosine determination in urine for prostatic carcinoma detection

Sarcosine, a potential biomarker for prostate cancer, can be detected in a solid state enzyme based biosensor using sarcosine oxidase, with particle immobilised reagents. A novel fusion protein of the fluorescent protein, mCherry, sarcosine oxidase (SOx), and the polypeptide R5 (R52-mCherry-SOx-R5-6 H), was explored, which allowed self-immobilization on silica microparticles and long-term (90 days +) retention of activity, even at room temperature. In contrast, commercial wildtype SOx lost activity in a few days. A silica-R52-mCherry-SOx-R5-6H microparticle sensor for determination of sarcosine in urine, linked the SOx coproduct, H2O2, to a measurement catalysed by horseradish peroxidase (HRP) immobilised on silica, in the presence of Amplex Ultrared (AR) to generate fluorescence at 582 nm. Silica microparticles carrying all the reagents (R52-mCherry-SOx-R5-6H, HRP and AR) were used to produce a silica-microparticle biosensor which responded to sarcosine at micromolar levels. Interference by amino acids and uric acid was examined and it was found that the silica-reagent carrying system could be calibrated in urine and responded across the clinically relevant concentration range. This contrasted with similar assays using commercial SOx, where interference inhibited the sarcosine signal measurement in urine. The microparticle biosensor was tested in urine from healthy volunteers and prostate cancer patients, showing higher concentrations of sarcosine in cancer patients consistent with previous reports of elevated sarcosine levels

Quantitative Analysis of Terpenic Compounds in Microsamples of Resins by Capillary Liquid Chromatography

A method has been developed for the separation and quantification of terpenic compounds typically used as markers in the chemical characterization of resins based on capillary liquid chromatography coupled to UV detection. The sample treatment, separation and detection conditions have been optimized in order to analyze compounds of different polarities and volatilities in a single chromatographic run. The monoterpene limonene and the triterpenes lupeol, lupenone, β-amyrin, and α-amyrin have been selected as model compounds. The proposed method provides linear responses and precision (expressed as relative standard deviations) of 0.6% to 17%, within the 0.5–10.0 µg mL−1 concentration interval; the limits of detection (LODs) and quantification (LOQs) were 0.1–0.25 µg mL−1 and 0.4–0.8 µg mL−1, respectively. The method has been applied to the quantification of the target compounds in microsamples. The reliability of the proposed conditions has been tested by analyzing three resins, white copal, copal in tears, and ocote tree resin. Percentages of the triterpenes in the range 0.010% to 0.16% were measured using sample amounts of 10–15 mg, whereas the most abundant compound limonene (≥0.93%) could be determined using 1 mg portions of the resins. The proposed method can be considered complementary to existing protocols aimed at establishing the chemical fingerprint of these kinds of samples