A Polypyrrole/Nanoclay Hybrid Film for Ultra-Sensitive Cardiac Troponin T Electrochemical Immunosensor.

An electrochemical immunosensor based on a nanohybrid film of carboxylated polypyrrole and amine nanoclay was developed for label-free detection of the human cardiac troponin T (cTnT). The nanohybrid film was formed in situ on the surface of the glassy carbon electrode, followed by the covalent immobilization of anti-troponin T antibodies by glutaraldehyde. Morphological and chemical characterizations of the nanohybrid film were performed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Under the optimized conditions, a calibration curve for cTnT in spiked serum was obtained by square wave voltammetry, and a low limit of detection and quantification was achieved (0.35 and 1.05 pg mL-1, respectively). This was the first time that this type of nanohybrid film was used in the development of an immunosensor for cTnT that proved to be a simple and efficient strategy for the manufacture of a label-free electrochemical device that could be applied in the diagnosis of acute myocardial infarction.

A Label and Probe-Free Zika Virus Immunosensor Prussian Blue@carbon Nanotube-Based for Amperometric Detection of the NS2B Protein.

Zika virus (ZIKV) is a mosquito-borne infection, predominant in tropical and subtropical regions causing international concern due to the ZIKV disease having been associated with congenital disabilities, especially microcephaly and other congenital abnormalities in the fetus and newborns. Development of strategies that minimize the devastating impact by monitoring and preventing ZIKV transmission through sexual intercourse, especially in pregnant women, since no vaccine is yet available for the prevention or treatment, is critically important. ZIKV infection is generally asymptomatic and cross-reactivity with dengue virus (DENV) is a global concern. An innovative screen-printed electrode (SPE) was developed for amperometric detection of the non-structural protein (NS2B) of ZIKV by exploring the intrinsic redox catalytic activity of Prussian blue (PB), incorporated into a carbon nanotube-polypyrrole composite. Thus, this immunosensor has the advantage of electrochemical detection without adding any redox-probe solution (probe-less detection), allowing a point-of-care diagnosis. It was responsive to serum samples of only ZIKV positive patients and non-responsive to negative ZIKV patients, even if the sample was DENV positive, indicating a possible differential diagnosis between them by NS2B. All samples used here were confirmed by CDC protocols, and immunosensor responses were also checked in the supernatant of C6/36 and in Vero cell cultures infected with ZIKV.

A probeless and label-free electrochemical immunosensor for cystatin C detection based on ferrocene functionalized-graphene platform.

A novel electrochemical sensor with inherent redox activity mediated by ferrocene for Cystatin C (CysC), an early kidney failure biomarker, is described. The current response was mediated by graphene oxide-ferrocene nanofilm with redox-activity coming from electroactive species surface-confined. Anti-CysC antibodies were immobilized by their Fc portions on the drop-casting polyethyleneimine (PEI) film for improving the sensitivity and reproducibility. Stepwise modifications of the nanostructured surface were characterized by electrochemical techniques, FT-IR and AFM. FT-IR confirmed the formation of the Fc-GO nanocomposite and PEI deposition on the electrode surface. The AFM micrographs confirmed a nanometric film of Fc-GO and PEI. The sensor platform showed a response from 0.1 to 1000 ng/mL and lower limit of detection (LOD) of 0.03 ng/mL of CysC, with good accuracy, specificity and it was successfully applied for CysC detection. Advantages of this immunosensor include rapid testing with minimal steps by the simple use of an intrinsic redox probe, working in a reduction potential, which avoids potential interferences. This proposal attempts to circumvent amperometric detection limitations and provides a promising candidate for future point-of-care diagnostics without redox probe additional solutions for measurements.

Cratylia mollis lectin nanoelectrode for differential diagnostic of prostate cancer and benign prostatic hyperplasia based on label-free detection.

The research for new biomarkers of cancer has studied the role of fetuin glycoprotein on the metastatic disease diagnosis. Cratylia mollis is a lectin with high finity to fetuin, and used here to differentiate prostate cancer and benign prostatic hyperplasia. A label-free electrochemical nanosensor based on assembled carboxylated carbon nanotubes (COOH-CNTs) and poly-L-lysine (PLL) film was developed and applied to serum samples of prostate cancer positive for Gleason score. The electrode analytical response to fetuin in PBS samples, obtained by square wave voltammetry, exhibited a linear range from 0.5 to 25µgmL(-1), with a high correlation coefficient (r=0.994, p<0.001) and low limit of detection (0.017µgmL(-1)). The lectin nanoelectrode showed a good repeatability (1.24% RSD) and reproducibility (4.24% RSD). A pool of serum samples from prostate cancer patients with known the Gleason score were tested showing a significant statistically correlation. Thus, the lectin nanoelectrode was able to distinguish the degree of staging prostate cancer, providing the diagnostic differentiation of benign and malign hyperplasia. To the best of our knowledge, it is the first biosensor for this application using a lectin.

An ultrasensitive human cardiac troponin T graphene screen-printed electrode based on electropolymerized-molecularly imprinted conducting polymer.

A nano-molecularly imprinted polymer (N-MIP) assembled on a screen-printed electrode for the cardiac troponin T (cTnT) was developed. The biomimetic surface was obtained by a co-polymer matrix assembled on the reduced graphene oxide (RGO) electrode surface. The cTnT active sites were engineered using pyrrole and carboxylated pyrrole that was one-step electropolymerized jointly with cTnT by cyclic voltammetry. The stepwise preparation of the biomimetic surface was characterized by cyclic and differential pulse voltammetries using the ferrocyanide/ferricyanide as redox probe. Structural and morphological characterization was also performed. The optimal relation of pyrrole and pyrrole-3-acid carboxylic to perform the cTnT biomimetic nanosurface was obtained at 1:5 ratio. The analytical performance of cTnT N-MIP performed by differential pulse voltammetry showed a linear range from 0.01 to 0.1 ngmL(-1) (r=0.995, p«0.01), with a very low limit of detection (0.006 ngmL(-1)). The synergic effect of conductive polymer and graphene forming 3D structures of reactive sites resulted in a N-MIP with excellent affinity to cTnT binding (KD=7.3 10(-13) molL(-1)). The N-MIP proposed is based on a simple method of antibody obtaining with a large potential for point-of-care testing applications.

Amino-functionalization of carbon nanotubes by using a factorial design: human cardiac troponin T immunosensing application.

A simple amino-functionalization method for carbon nanotubes and its application in an electrochemical immunosensor for detection of the human cardiac troponin T are described. Amino-functionalized carbon nanotubes allow oriented antibodies immobilization via their Fc regions, improving the performance of an immunosensor. Herein multiwalled carbon nanotubes were amino-functionalized by using the ethylenediamine reagent and assays were designed by fractional factorial study associated with Doehlert matrix. Structural modifications in the carbon nanotubes were confirmed by Fourier transform infrared spectroscopy. After amino-functionalization the carbon nanotubes were attached to screen-printed carbon electrode and a sandwich-type immunoassay was performed for measuring the cardiac troponin T. The electrochemical measurements were obtained through hydrogen peroxide reaction with peroxidase conjugated to the secondary antibody. Under optimal conditions, troponin T immunosensor was evaluated in serum samples, which showed a broad linear range (0.02 to 0.32 ng mL(-1)) and a low limit of detection, 0.016 ng mL(-1). This amino platform can be properly used as clinical tool for cardiac troponin T detection in the acute myocardial infarction diagnosis.

A carbon nanotube screen-printed electrode for label-free detection of the human cardiac troponin T.

Label-free immunosensor based on amine-functionalized carbon nanotubes screen-printed electrode is described for detection of the cardiac troponin T, an important marker of acute myocardial infarction. The disposable sensor was fabricated by tightly squeezing an adhesive carbon ink containing carbon nanotubes onto a polyethylene terephthalate substrate forming a thin film. The use of carbon nanotubes increased the reproducibility and stability of the sensor, and the amine groups permitted nonrandom immobilization of antibodies against cardiac troponin T. Amperometric responses were obtained by differential pulse voltammetry in presence of a ferrocyanide/ferricyanide redox probe after troponin T incubation. The calibration curve indicated a linear response of troponin T between 0.0025 ng mL(-1) and 0.5 ng mL(-1), with a good correlation coefficient (r=0.995; p<0.0001, n=7). The limit of detection (0.0035 ng mL(-1) cardiac troponin T) was lower than any previously described by immunosensors and was comparable with conventional analytical methods. The high reproducibility and clinical range obtained using this immunosensor support its utility as a potential tool for point-of-care acute myocardial infarction diagnostic testing.

Disposable immunosensor for human cardiac troponin T based on streptavidin-microsphere modified screen-printed electrode.

Screen-printed electrodes (SPE) have been widely used in the design of disposable sensors bringing advances in the use of electrochemical immunosensors for in field-clinical analysis. In this work, streptavidin polystyrene microspheres were incorporated to the electrode surface of SPEs in order to increase the analytical response of the cardiac troponin T (cTnT), a specific biomarker for the acute myocardial infarction diagnosis. The precise calculation of the stoichiometric streptavidin-biotin ratio [1:4] allowed the increase of sensitivity and stability of the immunosensor response to the cTnT analyte. The surface of the immunosensor was characterized by scanning electron microscopy and cyclic voltammetry. It was observed that the use of streptavidin microspheres significantly increased the analytical sensitivity of the electrode in 8.5 times, showing a curve with a linear response range between 0.1 and 10 ngmL(-1) of cTnT and a detection limit of 0.2 ngmL(-1). The proposed SPE showed ease preparation and high sensitivity allowing the detection of cTnT in the range of clinical levels. The new device coupled with a portable electrochemical analyzer shows great promise for point-of-care quantitative testing of necrosis cardiac proteins.