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Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element. Determination of fibrinogen in plasma.

Significance Statement

In this work two different amperometric immunosensing designs for the determination of fibrinogen using a novel and specific His-tagged nanobody expressed in Escherichia coli, functionalized magnetic beads and disposable screen-printed electrodes are described for the first time. Direct and indirect competitive magnetoimmunosensing configurations have been implemented and compared. In the direct one, fibrinogen and biotinylated fibrinogen competed for the binding sites of nanobodies immobilized on His-tag Isolation MBs while the indirect configuration involved competition of free fibrinogen in solution and fibrinogen immobilized on carboxylic acid modified MBs for a fixed amount of the specific biotinylated nanobody. In both configurations, the final labeling was made with Strep-HRP, the resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at -0.20 V (vs. a Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate.

Although both configurations would allow quantification of fibrinogen at the clinical relevant concentration by appropriate sample dilution, the indirect competitive configuration allowed a much better sensitivity. It is worth to mention that the LOD attained with the indirect competitive magnetoimmunosensor (0.044 mg mL-1) is 68,000 times lower than the normal fibrinogen concentration in plasma.

The usefulness of the developed indirect competitive magnetoimmunosensor was checked by analyzing an International Standard for fibrinogen Plasma containing a certified concentration of 2.7 mg mL-1. After evaluating the absence of matrix effect, the determination of fibrinogen was accomplished by interpolation of the amperometric values measured for the reconstituted plasma appropriately diluted into the corresponding calibration plot obtained with fibrinogen standard solutions. The statistical comparison between the obtained and the certified values demonstrated the absence of significant differences for a confidence level of 95%. It should be remarked that these satisfactory results implied a minimal sample treatment (just a dilution in the buffer solution) and a total assay time of 90 min (once the receptor modified MBs were prepared).

The great analytical performance exhibited the simplicity of the methodologies and the use of disposable mass-produced biosensors constitutes important advantages for their consideration as suitable methodologies for POC clinical diagnosis and prognosis.

 

Disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element. Determination of fibrinogen in plasma

 

Campuzano S, Salema V, Moreno-Guzmán M, Gamella M, Yáñez-Sedeño P, Fernández LA, Pingarrón JM.

Biosens Bioelectron. 2014 Feb 15;52:255-60.

Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain.

  

Abstract

 

Two different fibrinogen (Fib) amperometric immunosensing designs based on the use of magnetic beads (MBs) and a novel specific nanobody (Nb) expressed in Escherichia coli are described for the first time. The immunological reaction for Fib detection was performed on COOH-MBs or His-Tag-Isolation-MBs as solid support for the immobilization of the antigen or the captured Nb. Direct and indirect competitive magnetoimmunosensing configurations have been tested and compared. In the former one, Fib and biotinylated Fib competed for the immobilized Nb binding sites while the latter configuration involved competition of free Fib in solution and immobilized Fib for binding to a fixed amount of the specific biotinylated Nb. Labeling of the captured biotinylated Nb or antigen was made with streptavidin-HRP. The modified magnetic beads were captured by a neodymium magnet on the surface of screen-printed carbon electrodes (SPCEs). Amperometric detection was accomplished at -0.20 V (vs. Ag pseudo-reference electrode) by measuring the catalytic current arising upon addition of H2O2 and using hydroquinone (HQ) as redox mediator in solution. A better analytical performance was achieved with the indirect competitive immunoassay with a detection limit of 0.044 ug mL(-1) Fib. The usefulness of both approaches was successfully demonstrated by analyzing an international standard for Fib plasma. The assays could be carried out in diluted plasmasamples in a total analysis time of 90 min.

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