Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate -NO₂ groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC₅₀ values for atrazine and 2,4-D equal to 0.8 ng mL⁻¹ and 7 ng mL⁻¹, respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL⁻¹ respectively in the optimum working range between 0.01 and 1000 ng mL⁻¹ with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.     

Nanogold–polyaniline–nanogold microspheres-functionalized molecular tags for sensitive electrochemical immunoassay of thyroid-stimulating hormone

Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold–polyaniline–nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab₂) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L⁻¹ H₂O₂ by using GPG-labeled HRP-Ab₂ as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab₂. Several labeling protocols comprising HRP-Ab₂, nanogold particle-labeled HRP-Ab₂, and polyaniline nanospheres-labeled HRP-Ab₂, were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab₂. With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab₂ to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 μIU mL⁻¹ with a detection limit (LOD) of 0.005 μIU mL⁻¹ TSH at the 3s_B criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH.     

Multiplex detection of tumor markers with photonic suspension array

A novel photonic suspension array was developed for multiplex immunoassay. The carries of this array were silica colloidal crystal beads (SCCBs). The codes of these carriers are the characteristic reflection peak originated from their structural periodicity, and therefore they do not suffer from fading, bleaching, quenching, and chemical instability. In addition, because no dyes or materials related with fluorescence are included, the fluorescence background of SCCBs is very low. With a sandwich format, the proposed suspension array was used for simultaneous multiplex detection of tumor markers in one test tube. The results showed that the four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), carcinoma antigen 125 (CA 125) and carcinoma antigen 19-9 (CA 19-9) could be assayed in the ranges of 1.0-500 ng mL⁻¹, 1.0-500 ng mL⁻¹, 1.0-500 U mL⁻¹ and 3.0-500 U mL⁻¹ with limits of detection of 0.68 ng mL⁻¹, 0.95 ng mL⁻¹, 0.99 U mL⁻¹ and 2.30 U mL⁻¹ at 3σ, respectively. The proposed array showed acceptable accuracy, detection reproducibility, storage stability and the results obtained were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. This technique provides a new strategy for low cost, automated, and simultaneous multiplex immunoassay.     

Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategy

In this paper, a simple and sensitive amperometric immunosensor for simultaneous detection of four biomarkers by using distinguishable redox-probes as signal tags was proposed for the first time. In sandwich immunoassay format, four kinds of capture antibodies (C-Ab) were immobilized by gold nanoparticles (AuNPs) electro-deposited on the surface of glass carbon electrode (GCE); four kinds of detection antibodies (D-Ab) labeled with different redox probes (including anthraquinone 2-carboxylic acid (Aq), thionine (Thi), ferrocenecarboxylic acid (Fc) and tris(2,2’-bipyridine-4,4’-dicarboxylic acid) cobalt(III) (Co(bpy)₃³⁺)), were combined with 3,4,9,10-perylenetetracarboxylic acid (PTCA), poly(diallyldimethylammonium chloride) (PDDA) and AuNPs functionalized carbon nanotubes, and served as signal tracer. When four target antigens were present, differential pulse voltammetry (DPV) scan exhibited four well-resolved peaks, each peak indicated one antigen, and its intensity was quantitative correlational to the concentration of corresponding analyte. To verify the strategy, four biomarkers for diagnosis of colorectal carcinoma, including carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 CA125, and CA242, were used as model analytes, the immunosensor exhibited high selectivity and sensitivity, and peak current displayed good linear relationship to logarithm concentration in the ranges from 0.016 to 15 ng mL⁻¹ for CEA; 0.008 to 10 ng mL⁻¹ for CA19-9; 0.012 to 12 ng mL⁻¹ for CA125; 0.010 to 10 ng mL⁻¹ for CA242, and low detection limits of 4.2, 2.8, 3.3 and 3.8 pg mL⁻¹, respectively.     

An electrochemical immunosensor for carcinoembryonic antigen enhanced by self-assembled nanogold coatings on magnetic particles

A quick and reproducible electrochemical-based immunosensor technique, using magnetic core/shell particles that are coated with self-assembled multilayer of nanogold, has been developed. Magnetic particles that are structured from Au/Fe₃O₄ core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The carcinoembryonic antibody (anti-CEA) was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode (SPCE) by an external magnetic field. This is an assembly of a novel immuno biosensor for carcinoembryonic antigen (CEA). The sensitivity and response features of this immunoassay are significantly affected by the surface area and the biological compatibility of the multilayered nanogold. The linear range for the detection of CEA was from 0.005 to 50 ng mL⁻¹ and the limit of detection (LOD) was 0.001 ng mL⁻¹. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA detection.     

Multiplex immunodetection of tumor markers with a suspension array built upon core-shell structured functional fluorescence-encoded microspheres

A new suspension array built upon laboratory-prepared functional fluorescence-encoded polystyrene beads (FFPBs) was developed for multiplex immunodetection of tumor markers. The FFPBs were synthesized by copolymerizing rhodamine 6G (R6G) and carboxyl function groups on the surface of the seed beads forming a core-shell structure. The fabrication process was facile and the encoding fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. In present work, we demonstrated that the quantity variation of impregnated R6G had negligible effect on the coupling efficiency of biomolecules onto the surface of the FFPBs. The R6G encoding fluorescence remained good monodispersity upon capture probe coupling and immunocomplex formation. No fluorescence resonance energy transfer was observed between the R6G doped in the bead shell and fluorophore used for antibody labeling. Under the optimal conditions, the proposed suspension array allowed simultaneous detection of α-fetoprotein, carcinoembryonic antigen, and prostate specific antigen in the ranges of 0.07-500 ng mL⁻¹, 1-2000 ng mL⁻¹, and 0.5-500 ng mL⁻¹, respectively, with detection limits of 0.0626 ng mL⁻¹, 0.554 ng mL⁻¹, and 0.250 ng mL⁻¹. Test on clinical serum samples demonstrated that the results obtained with suspension array were in good agreement with those of the reference electrochemiluminescence immunoassay method. We conclude that the laboratory-made FFPBs are sufficient as the microcarrier for the construction of suspension array in clinical diagnosis.     

Immunochemical method for sulfasalazine determination in human plasma

Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized.The immunoassay showed a high sensitivity (IC₅₀ = 0.51 ng mL⁻¹) and specificity, a detection limit of 0.02 ng mL⁻¹ and a dynamic range of 0.06-3.75 ng mL⁻¹ (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL⁻¹) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV = 13%).Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses.     

Sensitive sandwich immunoassay based on single particle mode inductively coupled plasma mass spectrometry detection

A sensitive sandwich type immunoassay has been proposed with the detection by inductively coupled plasma mass spectrometry (ICP-MS) in a single particle mode (time resolved analysis). The signal induced by the flash of ions (¹⁹⁷Au⁺) due to the ionization of single Au-nanoparticle (Au-NP) label in the plasma torch can be measured by the mass spectrometer. The frequency of the transient signals is proportional to the concentration of Au-NPs labels. Characteristics of the signals obtained from Au-NPs of 20, 45 and 80 nm in diameters were discussed. The analytical figures for the determination of Au-labeled IgG using ICP-MS in conventional integral mode and single particle mode were compared in detail. Rabbit-anti-human IgG was used as a model analyte in the sandwich immunoassay. A detection limit (3σ) of 0.1 ng mL⁻¹ was obtained for rabbit-anti-human IgG after immunoreactions, with a linear range of 0.3-10 ng mL⁻¹ and a RSD of 8.1% (2.0 ng mL⁻¹). Finally, the proposed method was successfully applied to spiked rabbit-anti-human IgG samples and rabbit-anti-human serum samples. The method resulted to be a highly sensitive ICP-MS based sandwich type immunoassay.     

Flow-injection catalytic spectrophotometic determination of molybdenum(VI) in plants using bromate oxidative coupling of p-hydrazinobensenesulfonic acid with N-(1-naphthyl)ethylenediamine

A novel flow-injection spectrophotometry has been developed for the determination of molybdenum(VI) at nanograms per milliliter levels. The method is based on the catalytic effect of molybdenum(VI) on the bromate oxidative coupling of p-hydrazinobenzenesulfonic acid with N-(1-naphthyl)ethylenediamine to form an azo dye (λ_max = 530 nm). Chromotropic acid (4,5-dihydroxy-2,7-naphthalenedisulfonic acid) acted as an effective activator for the molybdenum(VI)-catalyzed reaction and increased the sensitivity of the method. The reaction was monitored by measuring the change in absorbance of the dye produced. The proposed method allowed the determination of molybdenum(VI) in the range 1.0-20 ng mL⁻¹ with sample throughput of 15 h⁻¹. The limit of detection was 0.5 ng mL⁻¹ and a relative standard deviation for 10 ng mL⁻¹ molybdenum(VI) (n = 10) was 2.5%. The interfering ions were eliminated by using the combination of a masking agent and on-line minicolumn packed with cation exchanger. The present method was successfully applied to the determination of molybdenum(VI) in plant foodstuffs.     

Development of an ICP-MS immunoassay for the detection of anti-erythropoietin antibodies

A sandwich-type immunoassay linked with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the detection of anti-erythropoietin antibodies (anti-EPO Abs). Recombinant human erythropoietin (rhEPO) was immobilized on the solid phase to capture anti-rhEPO Abs specifically. After the immunoreactions with Au-labeled goat-anti-rabbit IgG, a diluted HNO₃ (2%) was used to dissociate Au nanoparticles which was then introduced to the ICP-MS for measurements. Under the optimized conditions, the calibration graph for anti-EPO Abs was linear in the range of 35.6-500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹ (3σ, n = 9). The relative standard deviation (R.S.D.) for three replicate measurements of 30.9 ng mL⁻¹ of anti-EPO Abs was 8.43%. The recoveries of anti-EPO Abs in sera at the spiking level of 50, 100, 150, 200 and 400 ng mL⁻¹ were 99.2%, 101.5%, 95.0%, 94.0% and 102.9%, respectively. For the real sample analysis, 26 samples from healthy people and 53 samples from patients with rhEPO treatments were studied. One sample from patients showed significantly higher anti-EPO Abs from other samples, indicating a possibility of immune response of this patient.     

Production and characterization of a broad-specificity polyclonal antibody for O,O-diethyl organophosphorus pesticides and a quantitative structure-activity relationship study of antibody recognition

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphorothioyloxy)benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a heterologous coating antigen, 4-(3-(diethoxyphosphorothioyloxy)phenylamino)-4-oxobutanoic acid. The 50% inhibition value (IC₅₀) was 348 ng mL⁻¹ for parathion, 13 ng mL⁻¹ for coumaphos, 22 ng mL⁻¹ for quinalphos, 35 ng mL⁻¹ for triazophos, 751 ng mL⁻¹ for phorate, 850 ng mL⁻¹ for dichlofenthion, and 1301 ng mL⁻¹ for phoxim. The limit of detection (LOD) met the ideal detection criteria of all the seven OP residues. A quantitative structure-activity relationship (QSAR) model was constructed to study the mechanism of antibody recognition using multiple linear regression analysis. The results indicated that the frontier-orbital energies (energy of the highest occupied molecular orbital, E_HOMO, and energy of the lowest unoccupied molecular orbital, E_LUMO) and hydrophobicity (log of the octanol/water partition coefficient, log P) were mainly responsible for the antibody recognition. The linear equation was log(IC₅₀) = −63.274E_HOMO + 15.985E_LUMO + 0.556 log P − 25.015, with a determination coefficient (r²) of 0.908.     

A high-throughput method for determination of metabolites of organophosphate flame retardants in urine by ultra performance liquid chromatography–high resolution mass spectrometry

Organophosphate triesters are common flame retardants used in a wide variety of consumer products from which they can migrate and pollute the indoor environment. Humans may thus be continuously exposed to several organophosphate triesters which might be a risk for human health. An analytical method based on direct injection of 5 μL urine into an ultra performance liquid chromatography system coupled to a time-of-flight mass spectrometry has been developed and validated to monitor exposure to organophosphate triesters through their respective dialkyl and diaryl phosphate metabolites (DAPs). The targeted analytes were: di-n-butyl phosphate (DNBP), diphenyl phosphate (DPHP), bis(2-butoxyethyl) phosphate (BBOEP), bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2-propyl) phosphate (BCPP) and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP). Separation was achieved in less than 3 min on a short column with narrow diameter and small particle size (50 mm × 2.1 mm × 1.7 μm). Different mobile phases were explored to obtain optimal sensitivity. Acetonitrile/water buffered with 5 mM of ammonium hydroxide/ammonium formate (pH 9.2) was the preferred mobile phase. Quantification of DAPs was performed using deuterated analogues as internal standards in synthetic urine (averaged DAP accuracy was 101%; RSD 3%). Low method limits of quantification (MLQ) were obtained for DNBP (0.40 ng mL⁻¹), DPHP (0.10 ng mL⁻¹), BDCIPP (0.40 ng mL⁻¹) and BBOEP (0.60 ng mL⁻¹), but not for the most polar DAPs, BCEP (∼12 ng mL⁻¹) and BCPP (∼25 ng mL⁻¹). The feasibility of the method was tested on 84 morning urine samples from 42 mother and child pairs. Only DPHP was found above the MLQ in the urine samples with geometric mean (GM) concentrations of 1.1 ng mL⁻¹ and 0.57 ng mL⁻¹ for mothers and children respectively. BDCIPP was however, detected above the method limit of detection (MLD) with GM of 0.13 ng mL⁻¹ and 0.20 ng mL⁻¹. While occasionally detected, the GM of DNBP and BBOEP were below MLD in both groups.     

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF₆) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H₂O₂ catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL⁻¹ with a detection limit of 0.25 ng mL⁻¹. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.     

Palladium@gold bimetallic nanostructures as peroxidase mimic for development of sensitive fluoroimmunoassay

In this paper, gold nanoparticles coated with palladium dots (Pd@Au) bimetallic nanostructures have been reported to have a peroxidase like activity which is not found in their monometallic counterparts. Based on this finding, we have developed an immunoassay in which antibody-modified Pd@Au nanostructure catalyzes the dimerization of a fluorogenic substrate for peroxidase, 3-(4-dihydroxy phenyl) propionic acid (HPPA), to generate high fluorescence signal. Specific antibodies against bensulfuron-methyl were generated by using a well characterized bensulfuron–protein conjugate as an immunogen, and the assay was performed in a competitive immunoassay format where Pd@Au nanostructure was bound to secondary antibody to show the peroxidase like activity. The developed immunoassay exhibited an excellent sensitivity showing a dynamic response range from 0.001 to 100 ng mL⁻¹ for herbicide bensulfuron-methyl with a detection limit of 0.01 ng mL⁻¹ (n = 3). The newly synthesized bimetallic nanostructure shows the advantages of low cost, easy synthesis and tunable catalytic activity, making it a promising substitution of enzyme peroxidase in different applications.     

Synthesis and applications of surface-grafted Th(IV)-imprinted polymers for selective solid-phase extraction of thorium(IV)

A new functional monomer N-(o-carboxyphenyl)maleamic acid (CPMA) was synthesized and chosen for the preparation of surface-grafted ion-imprinted polymers (IIPs) specific for thorium(IV). Polymerizable double bond was introduced to silica gel surface by amidation reaction between -NH₂ and maleic anhydride. In the ion-imprinting process, thorium(IV) was complexed with the carboxyl groups, then was imprinted in the polymers grafted to the silica gel surface. The imprinted Th(IV) was removed with 3 mol L⁻¹ HCl. The obtained imprinted particles exhibited excellent selectivity and rapid kinetics process for Th(IV). The relatively selective factor (α_r) values of Th(IV)/La(III), Th(IV)/Ce(III), Th(IV)/Nd(III), Th(IV)/U(VI), and Th(IV)/Zr(IV) were 85.7, 88.9, 26.6, 64.4, and 433.8, respectively, which were greater than 1. The precision (R.S.D.), the detection limit (3σ), and the quantification limit (10σ) of the method were 1.9%, 0.51 ng mL⁻¹ and 1.19 ng mL⁻¹, respectively. The prepared IIPs as solid-phase extractants were successfully applied for the preconcentration of trace thorium in natural and certified samples prior to its determination by inductively coupled plasma atomic emission spectrometry (ICP-AES) with satisfactory results.     

Carbon monolith: Preparation,characterization and application as microextraction fiber

A carbon monolith was synthesized via a polymerization–carbonization method, styrene and divinylbenzene being adopted as precursors and dodecanol as a porogen during polymerization. The resultant monolith had bimodal porous substructure, narrowly distributed nano skeleton pores and uniform textural pores or throughpores. The carbon monolith was directly used as an extracting fiber, taking place of the coated silica fibers in commercially available solid-phase microextraction device, for the extraction of phenols followed by gas chromatography–mass spectrometry. Under the studied conditions, the calibration curves were linear from 0.5 to 50 ng mL⁻¹ for phenol, o-nitrophenol, 2,4-dichlorophenol and p-chlorophenol. The limits of detection were between 0.04 and 0.43 ng mL⁻¹. The recoveries of the phenols spiked in real water samples at 10 ng mL⁻¹ were between 85% and 98% with the relative standard deviations below 10%. Compared with the commercial coated ones (e.g. PDMS, CW/DVB and DVB/CAR/PDMS), the carbon monolith-based fiber had advantages of faster extraction equilibrium and higher extraction capacity due to the superior pore connectivity and pore openness resulting from its bimodal porous substructure.     

Ligandless-solidified floating organic drop microextraction method for the preconcentration of trace amount of cadmium in water samples

In this article, a new ligandless solidified floating organic drop microextraction (LL-SFODME) method has been developed for preconcentration of trace amount of cadmium as a prior step to its determination by flow injection-flame atomic absorption spectrometry (FI-FAAS). The methodology is based on the SFODME of cadmium with 1-dodecanol in the absence of chelating agent. Several factors affecting the microextraction efficiency, such as, pH, sodium dodecylbenzenesulfonate (SDBS) concentration, extraction time, stirring rate and temperature were investigated and optimized. Under optimized experimental conditions an enhancement factor of 205 was obtained for 100 mL of sample solution. The calibration graph was linear in the range of 1.0-25.0 ng mL⁻¹, the limit of detection (3s) was 0.21 ng mL⁻¹ and the limit of quantification (10s) was 0.62 ng mL⁻¹. The relative standard deviation (RSD) for 10 replicate measurements of 10 ng mL⁻¹ cadmium was 4.7%. The developed method was successfully applied to the extraction and determination of cadmium in standard and several water samples and satisfactory results were obtained.     

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts,utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10 min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78 ng mL⁻¹ and the CCβ to be 1 ng mL⁻¹. Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC–MS/MS showed a high correlation (R² = 0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1 ng mL⁻¹, and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1 ng mL⁻¹. This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1 μg L⁻¹. This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.     

A visual strip sensor for determination of iron

A visual strip has been developed for sensing iron in different aqueous samples like natural water and fruit juices. The sensor has been synthesized by UV-radiation induced graft polymerization of acrylamide monomer in microporous poly(propylene) base. For physical immobilization of iron selective reagent, the in situ polymerization of acrylamide has been carried out in the presence of 1,10-phenanthroline. The loaded strip on interaction with Fe(II) in aqueous solution turned into orange red color and the intensity of the color was found to be directly proportional to the amount of Fe(II) in the aqueous sample. The minimal sensor response with naked eye was found for 50 ng mL⁻¹ of Fe in 15 min of interaction. However, as low as 20 ng mL⁻¹ Fe could be quantified using a spectrophotometer. The detection limit calculated using the 3s/S criteria, where ‘s’ is the standard deviation of the absorbance of blank reagent loaded strip and ‘S’ is the slope of the linear calibration plot, was 1.0 ng mL⁻¹. The strip was applied to measure Fe in a variety of samples such as ground water and fruit juices.     

Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay

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