Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay.

The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

Ultra-sensitive electrochemical immunosensor using analyte peptidomimetics selected from phage display peptide libraries.

Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC(50) of 0.15 ngmL(-1) (linear range from 4.4 × 10(-3) to 10 ngmL(-1)). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9 × 1000 nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors.

Citrinin (CIT) determination in rice samples using a micro fluidic electrochemical immunosensor.

The development of an electrochemical immunosensor incorporated in a micro fluidic cell for quantification of citrinin (CIT) mycotoxin in rice samples is described for the first time. Both CIT present in rice samples and immobilized on a gold surface electrodeposited on a glassy carbon (GC) electrode modified with a cysteamine self-assembled monolayer were allowed to compete for the monoclonal mouse anti-CIT IgG antibody (mAb-CIT) present in solution. Then, an excess of rabbit anti mouse IgG (H+L) labelled with the horseradish peroxidase (secAb-HRP) was added, which reacts with the mAb-CIT which is in the immuno-complex formed with the immobilized CIT on the electrode surface. The HPR, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of catechol (H(2)Q) whose back electrochemical reduction was detected on a GC electrode at -0.15 V vs Ag/AgCl by amperometric measurements. The current measured is proportional to the enzymatic activity and inversely proportional to the amount of CIT present in the rice samples. This immunosensor for CIT showed a range of work between 0.5 and 50 ng mL(-1). The detection (LOD) and the quantification (LOQ) limits were 0.1 and 0.5 ng mL(-1), respectively. The coefficients of variation intra- and inter-assays were less than 6%. The electrochemical detection could be done within 2 min and the assay total time was 45 min. The immunosensor was provided to undertake at least 80 determinations for different samples with a minimum previous pre-treatment. Our electrochemical immunosensor showed a higher sensitivity and reduced analysis time compared to other analytical methods such as chromatographic methods. This methodology is fast, selective and very sensitive. Thus, the immunosensor showed to be a very useful tool to determine CIT in samples of cereals, mainly rice samples.

Determination of progesterone (P4) from bovine serum samples using a microfluidic immunosensor system.

Progesterone (P4) is a steroidal hormone with a vital role in the maintenance of human and animal health. This paper describes the development of an immunosensor coupled to glassy carbon (GC) electrode and integrated to a microfluidic system to quantify P4 from bovine serum samples in a fast and sensitive way. The serum samples spiked with a given P4 concentration and a given P4 concentration bound to horseradish peroxide (HPR) were simultaneously added and, therefore, they competed immunologically with sheep monoclonal anti-P4 antibodies that were immobilized at a rotating disk. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the chatecol (H(2)Q) oxidation to benzoquinone (Q). Its reverse electrochemical reduction to H(2)Q can be detected at a GC electrode surface at -0.15 V by chronoamperometric measurements. These current responses are proportional to the enzyme activity and inversely proportional to the P4 amount present in bovine serum samples. This P4 immunosensor showed a linear working range from 0.5 to 12.5 ng mL(-1). The detection (DL) and quantification (QL) limits were 0.2 and 0.5 ng mL(-1), respectively. The electrochemical immunosensor had a higher sensitivity than the ELISA method using conventional spectrophotometric detections. However, both methods allowed us to obtain similar detection limits. The immunosensor allowed us to make up to 100 determinations on different samples without any previous pre-treatment. This behavior proved to be suitable to detect P4 in routine veterinary, clinical, biological, physiological, and analytical assays.