Electrochemically oriented immobilization of antibody on poly-(2-cyano-ethylpyrrole)-coated gold electrode using a cyclic voltammetry

Using quartz crystal microbalance (QCM) as an immunosensor, this work investigates the contribution of a cyclic voltammetry (CV) on the proper immobilization of antibodies with the aim of enhancing its target recognition and binding ability. Primarily, CV in the range of −0.1 to 0.9 V was applied to form a layer of poly-(2-cyano-ethylpyrrole) (PCEPy) on gold quartz crystal electrode. Then the efficiencies of antibodies (anti-IgG, AIgG) immobilized electrochemically with CV applied in 0-0.65 V were compared to those immobilized via physical adsorption, by observing relative affinity towards AIgG-Fab and AIgG-Fc fragments. The results showed antibody-AIgG-Fab interaction could be enhanced about 4 times when CV is applied (11.2 ± 1.3 vs 41.6 ± 3.4 relative fluorescence unit). On the contrary, physisorbed antibodies showed a higher degree of affinity towards AIgG-Fc indicating inappropriate orientations of physisorbed antibodies. AIgG immobilized PCEPy-gold QC electrode was characterized further for its sensitivity towards a new target bovine albumin with both a QCM and fluorescence measurement. Such electrode exhibited a good sensitivity as well as a large linear dynamic range, from 0.4 μg/ml to 1.0 μg/ml and from 0.5 μg/ml to 10.0 μg/ml, at QCM and fluorescence measurement, respectively.     

A novel biosensing interfacial design based on the assembled multilayers of the oppositely charged polyelectrolytes

A novel biosensing interfacial design strategy has been produced by the alternate adsorption of the oppositely charged polyelectrolytes. A quartz-crystal microbalance (QCM) as a model transducer was modified by use of mercaptoacetic acid (MAA) self-assembled monolayer (SAM) and the adsorption multilayers of the oppositely charged polyelectrolytes. MAA-SAM was first applied to the gold electrode surface of the crystal, and the positively charged chitosan was used as a double-sided linker to attach the negatively charged alginate-HSA antibodies to the negatively charged MAA-SAM layer. The assembly process and conditions were studied using the real-time output device and the surface topologies of the resulting crystals were characterized by atomic force microscopy (AFM) imaging. It is discovered that the optimal pH of immobilizing antibodies was 7.2 and the suited dilution ratio of antibodies was 10:30. The proposed immunosensor in optimal conditions has a linear detection range of 12.3-184.5 μg/mL for HSA detection. Comparing with the direct immobilization method of antibodies, the immunosensor with the proposed immobilization procedure shows some advantages, such as improved sensitivity due to the well-retained antibody activity and the significantly extended detection range. In particular, the regeneration of the developed immunosensor was simple and fast. Analytical results indicate that the developed immobilization procedure is a promising alternative for the immobilization of biorecognition element on the electrode surface.     

Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls

Detection of pollutants is of significant importance for environmental protection. However, conventional monitoring methods are often time-consuming, and require expensive equipments. Biosensors based on enzyme linked immunosorbent assay (ELISA) provide an alternative method to conventional ones. In this research, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a micro-flow immunosensor chip. The immunosensor chips were fabricated by micro-electromechanical system (MEMS) technology. The quantitative determination of coplanar polychlorinated biphenyls (Co-PCBs) was performed by using a micro-flow immunosensor chip. Polystyrene beads were used as the solid substrate for the immobilization of Co-PCB antibody. The antibody-immobilized beads were introduced into the flow channel. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP) conjugated antigen, and non-HRP conjugated antigen was allowed to react in the flow channel. After the antigen-antibody reaction, addition of phosphate buffer solution containing hydrogen peroxide and the fluorogenic substrate produced a fluorescent dye, which was monitored with the resulting change in the fluorescence intensity. By using our micro-flow immunosensor chip, it was possible to determine the sensing range of Co-PCB derivatives up to 0.1 ppt in 30 s. This immunosensor chip had a wide linear range for Co-PCB detection from 0.1 pg/ml to 1.0 μg/ml. The regression analysis provided the correlation coefficients of r = 0.982−0.964 with good reproducibility and precision. In a series of five measurements with immunosensor chips prepared with a new batch of antibody-immobilized polystyrene beads, a relative standard deviation of 21.3% was obtained. Our immunosensor chip design reported here has the potential to be implemented to several different detection methodologies for numerous analytes.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Nanogold-functionalized magnetic beads with redox activity for sensitive electrochemical immunoassay of thyroid-stimulating hormone

A new electrochemical immunosensor for sensitive determination of thyroid-stimulating hormone (TSH) was designed by using redox-active nanogold-functionalized magnetic beads (GoldMag) as signal tags on the nanogold–graphene interface. To construct such GoldMag nanostructures, polyethyleneimine-functionalized magnetic beads (PEI-MBs) were initially prepared by using a wet chemical method, and the electroactive thionine molecules and gold nanoparticles were then alternately immobilized on the surface of PEI-MBs by using an opposite-charged adsorption technique and an in situ synthesis method, respectively. The synthesized GoldMag nanostructures were utilized as signal tags for the label of horseradish peroxidase-anti-TSH conjugates (HRP-anti-TSH). With a sandwich-type immunoassay format, the conjugated signal tags on the transducer were increased with the increasing TSH concentration in the sample, thus enhancing the signal of the electrochemical immunosensor due to the labeled HRP toward the catalytic reduction of H₂O₂. Under optimal conditions, the current was proportional to the logarithm of TSH concentration ranging from 0.01 to 20 μIU mL⁻¹ in pH 6.0 HAc–NaAc containing 6 mM H₂O₂. The detection limit (LOD) was 0.005 μIU mL⁻¹ TSH at 3s_B. The immunosensor displayed an acceptable reproducibility, stability and selectivity. In addition, the methodology was evaluated with human serum specimens, receiving good correlation with results from commercially available electrochemiluminescent analyzer.     

Enantiospecific resolution of rabeprazole by liquid chromatography on amylose-derived chiral stationary phase using photo diode array and polarimetric detectors in series

A simple and rapid liquid chromatographic method for enantioselective separation and determination of R-(+) and S-(−) enantiomers of rabeprazole in drugs and pharmaceuticals using photo diode array (PDA) and polarimetric detectors connected in series was developed. Chiralpak AD-H (250 mm × 4.6 mm) 5 μm column packed with amylose tris(3,5-dimethylphenyl carbamate) as a stationary phase and the mobile phase containing n-hexane:ethanol:2-propanol(75:15:10, v/v/v) in an isocratic mode has yielded baseline separation with resolution greater than 3.0 at 40 °C. Effects of ethanol, 2-propanol and temperature on separation were studied for optimum resolution. Lansoprazole sulphone was used as an internal standard (IS) for quantitative determination of individual enantiomers in bulk drugs as well as pharmaceutical formulations. The method was validated in terms of accuracy, precision and linearity according to ICH guidelines. The linearity of the method was studied in the range of 0.5-50 μg/ml and the r² was >0.9997. The inter- and intra-day precision of assay were determined (R.S.D. < 1%) and the recoveries were in the range of 99.63-100.22% with <1% R.S.D. The limits of detection (LOD) and quantification (LOQ) were 0.02 μg/ml and 0.07 μg/ml for both the enantiomers, respectively.     

Development of a chemiluminescent immunosensor for chloramphenicol

A direct competitive chemiluminescent immunosensor system that exploits the competition between chloramphenicol (CAP) as an analyte and CAP-horseradish peroxidase conjugate as a tracer for binding to an anti-CAP antibody on a solid support was devised by installing a flow-through cell which was connected to an injector and a peristaltic pump inside a dark box, followed by positioning a photomultiplier tube as light detector in front of it. The anti-CAP antibody was immobilized onto positively charged Biodyne B membrane pieces by a dipping procedure. The operating conditions for the immunosensor were selected with respect to substrate composition (0.25, 13.3 and 0.66 mM for luminol, H₂O₂ and p-iodophenol, respectively), injection volume of the substrate solution (200 μL) and the concentrations of antibody for immobilization (0.10 mg mL⁻¹) and tracer (0.030 mg mL⁻¹). At these conditions, sensor response according to analyte concentration was well fitted to a linear equation when plotted in semi-logarithmic scale, with the limit of detection for CAP of 10⁻⁸ M. By using the immunosensor, CAP measurement in the model samples prepared from five food materials was conducted.     

Hybrid mesoporous materials for on-line preconcentration of Cr(VI) followed by one-step scheme for elution and colorimetric determination at ultratrace levels

An hybrid mesoporous material synthesised in our laboratories for solid phase extraction (SPE) in flow through systems has been used for analytical purposes. The solid was obtained from mesoporous silica MCM-41 functionalized with 3-aminopropyltriethoxy silane by Sol-Gel methodology. In order to exploit the large sorption capacity of the material together with the possibility of modeling it for anions retention, a microcolumn (MC) filled with the solid was inserted in a flow system for preconcentration of Cr(VI) and its determination at ultratrace levels in natural waters. The analytical methodology involved a reverse flow injection system (rFI) holding a MC filled with the solid for the analyte extraction. Elution and colorimetric detection were carried out with 1-5 diphenylcarbazide (DPC) in sulfuric acid. DPC produced the reduction of Cr(VI) to Cr(III) together with the generation of a cationic red complex between Cr(III) and 1-5 diphenylcarbazone which was easily eluted and detected with a visible spectrophotometer. Moreover, the filling material got ready for the next sample loading remaining unspoiled for more than 300 cycles.The effect of several variables on the analytical signal as well as the influence of cationic and anionic interferences were discussed. Particular attention was given to sulfuric acid interference since it is the required media for the complex generation.Under optimal conditions, 99.8% of Cr(VI) recovery was obtained for a preconcentration time of 120 s (sample and DPC flow rates = 1 mL min⁻¹) and an elution volume of 250 μL. The limit of detection (3 s) was found to be 0.09 μg L⁻¹ Cr(VI) with a relative standard deviation (n = 10, 3 μg L⁻¹) of 1.8.Since no Cr(III) was retained by the solid material and Cr(VI) was completely adsorbed, electrothermal atomic absorption spectrometry (ET AAS) determinations of Cr(III) were also performed by simply measuring its concentration at the end of the microcolumn after Cr(VI) retention by the mesoporous solid.Applications to the determination of Cr(VI) and Cr(III) in natural waters and the validation of the methodology were also studied.     

Chemiluminescent optical fiber immunosensor for the detection of anti-West Nile virus IgG

An ELISA-based optical fiber methodology developed for the detection of anti-West Nile virus IgG antibodies in serum was compared to standard colorimetric and chemiluminescent ELISA based on microtiter plates. Colorimetric ELISA was the least sensitive, especially at high titer dilutions. The fiber-optic immunosensor based on the same ELISA immunological rationale was the most sensitive technique.     

Highly-sensitive label-free electrochemical carcinoembryonic antigen immunosensor based on a novel Au nanoparticles–graphene–chitosan nanocomposite cryogel electrode

For the first time, a simple and highly sensitive label-free electrochemical carcinoembryonic antigen (CEA) immunosensor based on a cryogel electrode has been developed and tested. The as-prepared nanocomposite combined the advantages of the graphene, AuNPs and chitosan (AuNPs–GP–CS) together with the ease of preparing a cryogel coupled to a silver deposition, to act as a redox mediator, on a Au electrode. Under the optimal conditions, the decrease of the cyclic voltammetry (CV) silver peak current was proportional to the CEA concentration over a range of from 1.0 × 10⁻⁶ to 1.0 ng mL⁻¹ with a detection limit of 2.0 × 10⁻⁷ ng mL⁻¹. This AuNPs–GP–CS cryogel electrode gave a 1.7 times higher sensitivity and 25 times lower detection limit than the non-cryogel electrode. Moreover, the proposed electrochemical immunosensor exhibited good selectivity, reproducibility and stability. When applied to analyse clinical serum samples, the data determined by the developed immunosensor were in agreement with those obtained by the current hospital analysis system (enzyme linked fluorescent assay) (P > 0.05), to indicate that the immunosensor would be potentially useful for clinical diagnostics.     

Reagentless amperometric carbohydrate antigen 19-9 immunosensor based on direct electrochemistry of immobilized horseradish peroxidase

A reagentless immunosensor for rapid determination of carbohydrate antigen 19-9 (CA19-9) in human serum was proposed. This strategy was based on the immobilization of antibody in colloidal gold nanoparticle modified carbon paste electrode and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to a CA19-9 antibody. The nanoparticles were efficient for preserving the activity of immobilized biomolecules. Thus, the immobilized HRP displayed its direct electrochemistry with a rate constant of 1.02 s⁻¹. The incubation of the immunosensor in phosphate buffer solution (PBS) including CA19-9 antigen leading to the formation of antigen-antibody complex, which made the block of electron transfer of HRP toward electrode and resulted in significant peak current decrease of HRP. Under the optimal conditions, the current decrease was proportional to CA19-9 concentrations ranging from 2 to 30 U/ml with a detection limit of 1.37 U/ml at a current decrease by 10%. The immunosensor showed an acceptable accuracy compared with those obtained from immunoradiometric assays, with intra-assay coefficient of 7.3 and 6.9% at CA19-9 concentrations of 5 and 15 U/ml, respectively, and inter-assay coefficient of 9.6% at a CA19-9 concentration of 20 U/ml. The storage stability was acceptable in a pH 7.0 PBS at 4 °C for 10 days. This method avoids the addition of electron transfer mediator, thus simplifies the immunoassay procedure and decreases the analytical time. It provides a new promising platform for clinical immunoassay.     

Optical fibre reflectance sensor for the determination and speciation analysis of iron in fresh and seawater samples coupled to a multisyringe flow injection system

A novel optical fibre reflectance sensor coupled to a multisyringe flow injection system (MSFIA) for the determination and speciation analysis of iron at trace level using chelating disks (iminodiacetic groups) is proposed. Once iron(III) has been retained onto a chelating disk, an ammonium thiocyanate stream is injected in order to form the iron(III)-thiocyanate complex which is spectrophotometrically detected at 480 nm. Iron(III) is eluted with 2 M hydrochloric acid so that the chelating disk is regenerated for subsequent experiments. The determination of total iron is achieved by the on-line oxidation of iron(II) to iron(III) with a suitable hydrogen peroxide stream.A mass calibration was feasible in the range from 0.001 to 0.25 μg. The detection limit (3s_b/S) was 0.001 μg. The repeatability (RSD), calculated from nine replicates using 1 ml injections of a 0.1 mg/l concentration, was 2.2%. The repeatability between five chelating disks was 3.6%. The applicability of the proposed methodology in fresh and seawater samples has been proved.The proposed technique has been validated by replicate analysis (n = 4) of certified reference materials of water with satisfactory results.     

Amberlite XAD-4 functionalized with succinic acid for the solid phase extractive preconcentration and separation of uranium(VI)

Amberlite XAD-4 resin has been functionalized with succinic acid by coupling it with dibromosuccinic acid after acetylation. The resulting resin has been characterized by FT-IR, elemental analysis and TGA and has been used for preconcentrative separation of uranium(VI) from host of other inorganic species prior to its determination by spectrophotometry. The optimum pH value for quantitative sorption of uranium(VI) in both batch and column modes is 4.5-8.0 and desorption can be achieved by using 5.0 ml of 1.0 mol l⁻¹ HCl. The sorption capacity of functionalized resin is 12.3 mg g⁻¹. Calibration graphs were rectilinear over the uranium(VI) concentrations in the range 5-200 μg l⁻¹. Five replicate determinations of 50 μg of uranium(VI) present in 1000 ml of solution gave a mean absorbance of 0.10 with a relative standard deviation of 2.56%. The detection limit corresponding to three times the standard deviation of the blank was found to be 2 μg l⁻¹. Various cationic and anionic species at 200-fold amounts do not interfere during the preconcentration of 5.0 μg of uranium(VI) present in 1000 ml (batch) or 100 ml (column) of sample solution. Further, adsorption kinetic and isotherm studies were also carried out by a batch method to understand the nature of sorption of uranium(VI) with the succinic acid functionalized resin. The accuracy of the developed solid phase extractive preconcentration method in conjunction with Arsenazo III procedure was tested by analyzing marine sediment (MESS-3) and soil (IAEA soil-7) reference material. Further, the above procedure has been successfully employed for the analysis of soil and sediment samples.     

Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I₅₀ and detection limits were 0.05-2.5 and 0.1-7.5 μg L⁻¹, 0.35 (±0.04) μg L⁻¹ and 0.9 (±0.1) μg L⁻¹, 60 and 100 μg L⁻¹ in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I₅₀ in real samples was 0.2 μg L⁻¹ corresponding to 1.6 μg/kg in the wheat sample with a detection limit of 0.4 μg/kg (calculated as blank signal −3σ). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r = 0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.     

Determination of cefuroxime axetil in tablets and biological fluids using liquid chromatography and flow injection analysis

In the present work, the separations of calixarene derivatives have been investigated using both high-performance liquid chromatography (HPLC) and nonaqueous capillary electrophoresis (NACE) techniques. HPLC-1 method with LC-318 (pore size = 300 Å) column and MeCN mobile phase was optimized for the separation of calixarenes. At the flow-rate of 1 ml/min p-nitrocalix[6]arene, calix[4]arene and calix[6]arene could be well baseline and symmetrically separated within 5 min. For the separation of p-tert-butylcalix[n]arenes (n = 4, 6, 8), HPLC-2 and NACE methods have been optimized. The optimal conditions in HPLC-2 method included NH₂ column and MeCN mobile phase, and p-tert-butylcalix[n]arenes (n = 4, 6, 8) were baseline separated within 10 min at 0.8 min/min. The optimal conditions for NACE method employed MeCN-H₂O (8:2, v/v) as the nonaqueous medium and 120 mM Tris/HCl (pH 9.0) as the buffer, and p-tert-butylcalix[n]arenes (n = 4, 6, 8) were successfully baseline resolved within 16 min. With the detection at 280 nm, the calibration lines were linear in the ranges of 1-200 μg/ml for calixarene derivatives by HPLC-1 and HPLC-2 methods, and of 2.5-200 μg/ml for p-tert-butylcalix[n]arenes (n = 4, 6, 8) by NACE method, respectively. The detection limits (S/N = 3) and recoveries ranged from 0.5 to 1.4 μg/ml and from 98.1 to 102.4% by both HPLC-1 and HPLC-2 methods, and from 1.3 to 2.0 μg/ml and from 97.9 to 105.1% by NACE method, respectively. The intra-day reproducibility of the methods was determined with satisfactory results. The proposed HPLC and NACE methods were accurate and reproducible, and could be utilized to separate and determine calixarene derivatives.     

Nanosilver-doped DNA polyion complex membrane for electrochemical immunoassay of carcinoembryonic antigen using nanogold-labeled secondary antibodies

A simple and sensitive electrochemical immunoassay protocol was developed for the detection of carcinoembryonic antigen (CEA) using nanosilver-doped DNA polyion complex membrane (PIC) as sensing interface. To construct such an immunosensor, double-stranded DNA was initially assembled onto the surface of thionine/Nafion-modified screen-printed carbon electrode to adsorb silver ions with positive charges, then silver ions were reduced to nanosilver particles with the aid of NaBH₄, and then anti-CEA antibodies were immobilized on the nanosilver surface. Gold nanoparticles conjugated with horseradish peroxidase-labeled anti-CEA were employed as signal antibodies for the detection of CEA with a sandwich-type assay format. Under optimal conditions, the immunosensor exhibited a dynamic range of 0.03-32 ng mL⁻¹ with a low detection limit of 10 pg mL⁻¹ CEA. Intra- and inter-assay imprecision (CVs) were <9.5% and 6.5%, respectively. The response could remain 90.1% of the original current at 30th day. 50 real samples were evaluated using the immunosensor and the enzyme-linked immunosorbent assay, respectively, and received in accordance with those two methods.     

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 μg l⁻¹ for carbaryl and TCP, respectively. The sensitivity attained (I₅₀ value) was around 30 μg l⁻¹ for both compounds. Linear working ranges were 15-53 μg l⁻¹ for carbaryl and 13-83 μg l⁻¹ for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.     

Determination of nucleotides,nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography–mass spectrometry

An ion-pairing reversed-phase liquid chromatography–mass spectrometry (IP-RP-LC–MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25 mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5′-monophosphate (UMP, 0.638–10.200 μg/mL), adenosine-5′-monophosphate (AMP, 0.24–7.80 μg/mL) and guanosine-5′-monophosphate (GMP, 0.42–13.50 μg/mL), seven nucleosides including adenosine (0.55–8.85 μg/mL), guanosine (0.42–6.75 μg/mL), uridine (0.33–10.50 μg/mL), inosine (0.21–6.60 μg/mL), cytidine (0.48–15.30 μg/mL), thymidine (0.20–6.30 μg/mL) and cordycepin (0.09–1.50 μg/mL), as well as six nucleobases, adenine (0.22–6.90 μg/mL), guanine (0.26–4.20 μg/mL), uracil (0.38–12.15 μg/mL), hypoxanthine (0.13–4.20 μg/mL), cytosine (0.39–12.45 μg/mL) and thymine (0.26–8.25 μg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01–0.16 μg/mL and 0.04–0.41 μg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24 h ambient temperature water immersion (ATWE) and 56 h ATWE extracts. Two transformation pathways including UMP → uridine → uracil and GMP → guanosine → guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP → adenosine → inosine → hypoxanthine was proposed in natural C. sinensis, while AMP → adenosine → adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis.     

Novel, simple and low-cost alternative method for fabrication of paper-based microfluidics by wax dipping

Paper-based microfluidic devices are an alternative technology for fabricating simple, low-cost, portable and disposable platforms for clinical diagnosis. Hereby, a novel wax dipping method for fabricating paper-based microfluidic devices (μPADs) is reported. The iron mould for wax dipping was created by a laser cutting technique. The designed pattern was transferred onto paper by dipping an assembly mould into melted wax. The optimal melting temperature and dipping time were investigated. The optimal melting temperature was in the range of 120-130 °C, and the optimal dipping time was 1 s. The whole fabrication process could be finished within 1 min without the use of complicated instruments or organic solvents. The smallest hydrophilic channel that could be created by the wax dipping method was 639 ± 7 μm in size. The reproducibility of the μPAD fabrication for hydrophilic channel width of the test zone and sample zone was 1.48% and 6.30%, respectively. To verify the performance of the μPAD, multiple colorimetric assays for simultaneous detection of glucose and protein in real samples were performed. An enzymatic assay and the bromocresol green (BCG) method were conducted on the paper device to determine the presence of glucose and protein in a test solution. The results of the assays were not significantly different from those of the conventional methods (p > 0.05, pair t-test and one-way ANOVA method). The wax dipping provides a new alternative method for fabricating lab-on-paper devices for multiple clinical diagnostics and will be very beneficial for developing countries.     

Enzyme inhibition-based biosensor for the electrochemical detection of microcystins in natural blooms of cyanobacteria

An electrochemical biosensor for the detection of microcystin has been developed based on the inhibition of the protein phosphatase 2A (PP2A) by this cyanobacterial toxin. The enzyme has been immobilised by entrapment using a poly(vinyl alcohol) azide-unit pendant water-soluble photopolymer (PVA-AWP). Electrode supports and immobilisation conditions have been optimised by colorimetric assays, the highest immobilisation yields being obtained with screen-printed graphite electrodes and the 1:2 PP2A:PVA ratio. Catechyl monophosphate (CMP), α-naphthyl phosphate (α-NP) and 4-methylumbelliferyl phosphate (4-MUP) have been used as phosphorylated substrates to monitor the protein phosphatase activity by electrochemical methods, the former providing the highest chronoamperometric currents at appropriate working potentials (+450 mV versus Ag/AgCl). Incubation with standard microcystin solutions has demonstrated the inhibition of the immobilised enzyme, proportional to the toxin concentration. The standard inhibition curve has provided a 50% inhibition coefficient (IC₅₀) of 83 μg L⁻¹, a limit of detection (LOD; 35% inhibition) of 37 μg L⁻¹, and 100% inhibition at about 1000 μg L⁻¹. Real samples of cyanobacterial blooms from the Tarn River (Midi-Pyrénées, France) have been analysed using the developed amperometric biosensor and the toxin contents have been compared to those obtained by a conventional colorimetric protein phosphatase inhibition (PPI) assay and high-performance liquid chromatography (HPLC). The results clearly justify the use of the developed amperometric biosensor as screening method for microcystin detection.