Electrochemical magnetoimmunosensor for the ultrasensitive determination of interleukin-6 in saliva and urine using poly-HRP streptavidin conjugates as labels for signal amplification

Abstract

A novel magnetoimmunosensor design for interleukin-6 (IL-6) which involved the covalent immobilization of anti-IL-6 antibodies onto carboxyl-functionalized magnetic microparticles and a sandwich-type immunoassay with signal amplification using poly-HRP-streptavidin conjugates is reported. All the variables concerning the preparation and the electroanalytical performance of the immunosensor were optimized. The use of poly-HRP-strept conjugates as enzymatic labels instead of conventional HRP-strept allowed enhanced signal-to-blank current ratios to be obtained. A linear calibration plot between the measured steady-state current and the log of IL-6 concentration was achieved in the 1.75 to 500 pg/mL range, which was not feasible when using HRP-strep as label. A limit of detection of 0.39 pg/mL IL-6 was obtained. The anti-IL-6-MB conjugates exhibited an excellent storage stability providing amperometric responses with no significant loss during at least 36 days. The magnetoimmunosensor showed also an excellent selectivity against potentially interfering substances. The immunosensor was used to determine IL-6 in urine samples spiked at three different concentration levels with clinical relevance. Moreover, IL-6 was measured in three different saliva samples corresponding to a periodontitis patient, a smoker volunteer, and a non-smoker volunteer. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.

Electrochemical immunosensor for sensitive determination of transforming growth factor (TGF) - β1 in urine

“…In order to get the highest possible sensitivity to achieve the goal mentioned in the Introduction for this work, an electrochemical signal amplification strategy was implemented by labeling the detection antibody with a peroxidase-streptavidin polymer instead of conventional HRP-Strept. This strategy has demonstrated to enhance sensitivity in the preparation of electrochemical immunosensors since multiple HRP molecules are available to be used in the biocatalysis of the enzyme substrate (Ojeda et al, 2014). The foreseen amplification effect provoked by the use of poly-HRP-Strept was confirmed by comparing the responses obtained with both conjugates.…”

“…Capture antibodies immobilization was followed by a blocking step with ethanolamine (step 2) and the implementation of a sandwich-type immunoassay employing a biotinylated secondary antibody and poly-HRP-Strept for signal amplification (step 3). The use of poly-HRP-Strept instead of conventional HRP-Strept conjugate has demonstrated to be advantageous for the design of sensitive electrochemical immunosensors as multiple HRP molecules are available to biocatalyze H 2 O 2 reduction (Ojeda et al, 2014). Once the MBs bearing the immuno-conjugates were transferred to the surface of SPCE, hydrogen peroxide was added (step 4) and the amperometric response at À200 mV in the presence of hydroquinone was measured according to the reactions displayed in Fig.…”

“…Ethanolamine was chosen as the blocking agent to minimize unspecific adsorptions onto HOOC-MBs after anti-TGF binding due to its proved efficiency for this purpose (Ojeda et al, 2014). In order to optimize this blocking step, 1 and 2 M ethanolamine solutions and different incubation times over 30-90 min range were tested.…”

Species-specific optical genosensors for the detection of mycotoxigenic Fusarium fungi in food samples

“…The use of enzymatic detection allows signal amplification since each HRP molecule can catalyze many cycles of conversions of substrate molecules to the fluorescent product, leading to an increment of the analytical signal. Moreover, the use of SA-poly-HRP as label further increases the amount of fluorescent molecules produced per binding event further enhancing assay sensitivity, as described before [51].…”

Streptavidin and gold nanoparticles-based dual signal amplification for sensitive magnetoelastic sensing of mercury using a specific aptamer probe

“…Streptavidin (SA) is a well-studied protein that consists of four subunits, each of which is capable of binding one molecule of biotin with very high affinity (Kd = 10 -15 M) [24]. This fact has been exploited to devise powerful and widely used tools for affinity chromatography, surface plasmon resonance biosensor and other analytical applications [25][26][27][28]. Gold nanoparticles (AuNPs) have been used as an amplifier in quartz crystal microbalance (QCM) detection technology [29].…”