A capillary-based amperometric flow immunoassay for 2,4,6-trichlorophenol

This paper describes the development of two different capillary-based heterogeneous competitive flow immunoassay formats (capillary flow injection immunoassay (CFIIA) and capillary sequential injection immunoassay (CSIIA)) for the determination of 2,4,6-trichlorophenol (2,4,6-TCP). The assays are based on the competition between the analyte and an analyte derivative labelled with the enzyme beta-galactosidase, for an anti-TCP antibody, followed by the injection of the mixture at equilibrium into a flow stream, where separation between the fractions bound and unbound to the antibody is performed in a glass capillary containing immobilised protein A. The antibody-tracer fraction retained inside the protein A capillary was measured by injection of 4-aminophenyl- beta- D-galactoside (4-APG), followed by amperometric detection of the enzymatically generated 4-aminophenol (4-AP), leading to a negative correlation between the signal and the analyte concentration.The two immunoassay formats were compared in terms of sensitivity and speed, giving IC(50) values of 1.41+/-0.03 and 1.64+/-0.07 micro g L(-1), detection limits of 0.2 and 0.4 micro g L(-1), and sample throughputs of 6 and 4 h(-1) for the CFIIA and CSIIA system, respectively.The influence of different interfering chlorophenolic compounds in the assay was minor, with only one exception (i.e. 2,4-dichlorophenol). In addition, different water matrices were tested (surface, tap, and rain water), showing that the matrix influence was negligible, except for rainwater, which resulted in a 30% increase in sensitivity. As a conclusion, the assay is suitable for the fast screening of TCP present at low concentration levels in water samples.     

Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at −0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.     

A highly sensitive microRNA biosensor based on ruthenium oxide nanoparticle-initiated polymerization of aniline

Well-defined voltammetric current peaks of polyaniline were observed at biosensors hybridized with ruthenium oxide nanoparticle-tagged microRNAs and incubated in a mixture of aniline/H(2)O(2), which can be used for direct microRNA expression profiling with excellent sensitivity.     

A label-free and self-assembled electrochemical biosensor for highly sensitive detection of cyclic diguanylate monophosphate (c-di-GMP) based on RNA riboswitch

Cyclic diguanylate monophosphate (c-di-GMP) is an important second messenger that regulates a variety of complex physiological processes involved in motility, virulence, biofilm formation and cell cycle progression in several bacteria. Herein we report a simple label-free and self-assembled RNA riboswitch-based biosensor for sensitive and selective detection of c-di-GMP. The detectable concentration range of c-di-GMP is from 50 nM to 1 μM with a detection limit of 50 nM.     

Streptavidin-enhanced surface plasmon resonance biosensor for highly sensitive and specific detection of microRNA

We are presenting a method for sensitive and specific detection of microRNA (miRNA) using surface plasmon resonance. A thiolated capture DNA probe with a short complete complementary sequence was immobilized on the gold surface of the sensor to recognize the part sequence of target miRNA, and then an oligonucleotide probe linked to streptavidin was employed to bind the another section of the target. The use of the streptavidin-oligonucleotide complex caused a ~5-fold increase in signal, improved the detection sensitivity by a factor of ~24, and lowered the detection limit to 1.7 fmol of miR-122. This specificity allowed a single mismatch in the target miRNA to be discriminated. The whole assay takes 30 min, and the surface of the sensor can be regenerated at least 30 times without loss in performance. The method was successfully applied to the determination of miRNA spiked into human total RNA samples.

A highly sensitive and rapid organophosphate biosensor based on enhancement of CdS-decorated graphene nanocomposite

Benzoyl peroxide (BPO) as a brightener is often added to wheat flour, and excessive use of this food additive is receiving increasing concern. Herein, a simple and fast method for fluorescence detection of BPO is proposed based on consecutive chemical reactions. In this approach, BPO first oxidizes Fe(2+) into Fe(3+) and the resulting Fe(3+) then induces the opening of the spirolactam ring of a new rhodamine derivative, N-methoxy rhodamine-6G spirolactam, switching on fluorescence of the detection system. More importantly, the fluorescence response of the reaction system to BPO is rather rapid and sensitive, with a detection limit of 6 mg kg(-1) (k=3), which makes it to be of great potential use in food safety analysis. The applicability of the proposed method has been successfully demonstrated on the determination of BPO in wheat flour samples.     

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25 ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45 ng/mL and the LOD can reach an ultralow level of 0.03 ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)₆]^3−/4−. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.     

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cows milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL^–1 and a quantification limit (LOQ) of only 2.0 pg mL^–1. These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5–15 ng mL^–1. Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL^–1 depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL^–1 for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.Dedicated to the memory of Wilhelm Fresenius.     

Biosensors based on highly sensitive acetylcholinesterases for enhanced carbamate insecticides detection

This paper presents the construction of amperometric biosensors for the highly sensitive detection of carbamate insecticides based on the inhibition of acetylcholinesterase (AChE). This enzyme was immobilised by entrapment in an optimised sol-gel matrix on TCNQ-modified screen-printed electrodes. The enzyme activity was estimated by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine using TCNQ as mediator. Wild and genetically engineered AChEs from Drosophila melanogaster (Dm) were chosen for their high sensitivity towards insecticides, which substantially improves the LOD compared with cholinesterases from other sources. The wild type and three mutant enzymes were tested against three carbamate insecticides: carbaryl, carbofuran and pirimicard. The best LOD were obtained with the Y370A mutant for carbaryl (1 × 10⁻⁸ M), the E69W mutant for pirimicarb (2 × 10⁻⁸ M) and the I161V mutant for carbofuran (8 × 10⁻¹⁰ M). The biosensors were applied to the analysis of two potable water samples.     

A highly sensitive detection platform based on surface-enhanced Raman scattering for Escherichia coli enumeration

A very sensitive and highly specific heterogeneous immunoassay system, based on surface-enhanced Raman scattering (SERS) and gold nanoparticles, was developed for the detection of bacteria and other pathogens. Two different types of gold nanoparticles (citrate-stabilized gold nanosphere and hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorod particles) were examined and this immunoassay was applied for the detection of Escherichia coli. Raman labels were constructed by using these spherical and rod-shaped gold nanoparticles which were first coated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and subsequently with a molecular recognizer. The working curve was obtained by plotting the intensity of the SERS signal of the symmetric NO₂ stretching of DTNB at 1,333 cm⁻¹ versus the concentration of the E. coli. The analytical performance of gold particles was evaluated via a sandwich immunoassay, and linear calibration graphs were obtained in the E. coli concentration range of 10¹–10⁵ cfu/mL with a 60-s accumulation time. The sensitivity of the Raman label fabricated with gold nanorods was more than three times higher than spherical gold nanoparticles. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the developed immunoassay to detect E. coli in real water samples was also demonstrated.     

A very sensitive flow system for the direct determination of copper in natural waters based on spectrophotometric detection

A very sensitive flow injection method with spectrophotometric detection has been developed for the on-line determination of copper in natural waters. The method exhibits a limit of detection three times lower than the most sensitive direct spectrophotometric method previously described and then allows the direct and simple in situ determination of copper in most natural waters.The method was based on the measurement of the absorbance of the coloured complex formed by copper with the chromogenic reagent di-2-pyridyl ketone benzoylhydrazone (dPKBH) in an alkaline medium. This complex presents stoichiometry 1:2 (Cu:dPKBH), and exhibits maximum absorbance at 370 nm. The manifold used was very simple, and consisted of two channels. The first one contained the sample while the second one contained the colorimetric reagent (3.3×10⁻⁴ M dPKBH in 10% ethanol), in a 1.6×10⁻² M phosphate buffer solution at pH 8. The performance of the system was optimised by using both univariate and modified simplex methodologies. When modified simplex was used, the best signal was obtained for a sample injection volume of 529 μl, a reaction coil length of 1.29 m, and a reagent flow rate of 4.8 ml min⁻¹. Under optimum conditions, the response was linear up to 3 mg l⁻¹ copper, the equation of the straight line being y=0.314x+5.2×10⁻⁴ (r²=0.998). The method allowed a sampling frequency of 40 samples per hour and exhibited a precision of 2.11% (as R.S.D., n=11). The limit of detection was 4.6 μg l⁻¹ (calculated as 3s_b/m, where s_b is the standard deviation of the y-intercept and m represents the slope of the straight line), and was therefore more sensitive than all the direct continuous methods reported previously.The method was successfully applied to the analysis of real water samples, with an average relative error of 5.32%.     

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.     

Development and evaluation of a highly sensitive rapid response enzymatic nanointerfaced biosensor for detection of putrescine

Putrescine (1,4-diaminobutane) a biologically active diamine has been found to be a valuable analyte for several clinical and analytical purposes. The present work deals with diamine oxidase immobilized on iron oxide nanoparticles for quantifying the amount of putrescine produced, by the decarboxylation of ornithine, which is converted into hydrogen peroxide by the enzyme diamine oxidase (DAO). This reaction can be quantified using electrochemical techniques, which forms the basis of this work. Iron oxide (Fe(3)O(4)) nanoparticles, synthesized using thermal co-precipitation, were chosen for immobilization of DAO due to its simple preparation procedure, high surface area and cost-effectiveness. The size of the particles was in the range of 25-35 nm and the enzyme was linked covalently by carbodiimide activation and confirmed using FT-IR. For detecting the hydrogen peroxide released in the reaction, a glassy carbon-working electrode coated with enzyme linked iron oxide nanoparticles was poised at +0.4 V versus an Ag/AgCl reference electrode and a platinum wire was used as the counter electrode. A step-wise increase in current was observed and linearity was obtained in the range of 2-8 nM, with 0.65 nM as the minimum detection limit and the response time was found to be 0.3 seconds. Ascorbic acid, a common interfering molecule in biological samples, did not interfere with the measurements indicating the high degree of specificity of the diamine oxidase-based nano-interfaced biosensor.     

A new strategy for highly sensitive immunoassay based on single-particle mode detection by inductively coupled plasma mass spectrometry

A highly sensitive immunoassay is proposed based on time-resolved inductively coupled plasma mass spectrometry with nanoparticles as tags to antibody. Instead of using traditional integral mode detection, the transient signals induced by the flash of ions in the plasma torch from the ionization of nanoparticles tagged on antibody were recorded in a time-resolved mode. Since, under certain conditions, the frequency of transient signals is directly correlated to the concentration of nanoparticle tags, the concentration of nanoparticle-tagged antibodies can be quantified by the frequency of transient signals. With the present instrument setup, gold nanoparticle (Au-NP) tags, as small as about 15 nm in diameter, can be detected. This protocol is evaluated for a competitive immunoassay and the linear range for α-fetoprotein is 0. 016–6. 8 µg/L (between 20 and 80% inhibition). The limit of quantification is 0. 016 µg/L (20% inhibition, IC₂₀) with a relative standard deviation of 4.2% (20% inhibition, 4 replicates) for α-fetoprotein. The present strategy provides a sensitive readout method for nanoparticle tags, which is quite promising for numerous applications in immunoassay, DNA hybridization, and other biological analyses.