An ImmunoFET Coupled with an Immunomagnetic Preconcentration Technique for the Sensitive EIS Detection of HF Biomarkers.

We propose a new strategy using a sandwich approach for the detection of two HF biomarkers: tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). For this purpose, magnetic nanoparticles (MNPs) (MNPs@aminodextran) were biofunctionalized with monoclonal antibodies (mAbs) using bis (sulfosuccinimidyl) suberate (BS3) as a cross-linker for the pre-concentration of two biomarkers (TNF-α and IL-10). In addition, our ISFETs were biofunctionalized with polyclonal antibodies (pAbs) (TNF-α and IL-10). The biorecognition between pAbs immobilized on the ISFET and the pre-concentrate antigen (Ag) on MNPs was monitored using electrochemical impedance spectroscopy (EIS). Our developed ImmunoFET showed a low detection limit (0.03 pg/mL) toward our target analyte when compared to previously published electrochemical immunosensors. It showed a higher sensitivity than for other HF biomarkers. Finally, the standard addition method was used to determine the unknown concentration in artificial saliva. The results matched with the expected values well.

Immuno field-effect transistor (ImmunoFET) for detection of salivary cortisol using potentiometric and impedance spectroscopy for monitoring heart failure.

Cortisol, a steroid hormone mostly known as "the stress hormone," plays many essential functions in humans due its involvement in several metabolic pathways. It is well-known that cortisol dysregulation is implied in evolution and progression of several chronic pathologies, including cardiac diseases such as heart failure (HF). However, although several sensors have been proposed to date for the determination of cortisol, none of them has been designed for its determination in saliva in order to monitor HF progression. In this work, a silicon nitride based Immuno field-effect transistor (ImmunoFET) has been proposed to quantify salivary cortisol for HF monitoring. Sensitive biological element was represented by anti-cortisol antibody bound onto the ISFET gate via 11-triethoxysilyl undecanal (TESUD) by vapor-phase method. Potentiometric and electrochemical impedance spectroscopy (EIS) measurements were carried out for preliminary investigations on device responsiveness. Subsequently, a more sensitive detection was obtained using electrochemical EIS. The proposed device has proven to have a linear response (R2 always >0.99), to be sensitive (with a limit of detection, LoD, of 0.005 ± 0.002 ng/mL), selective in case of other HF biomarkers (e.g. N-terminal pro B-type natriuretic peptide (NT-proBNP), tumor necrosis factor-alpha (TNF-α), and interleukin 10 (IL-10)), and accurate in cortisol quantification in saliva sample by performing the standard addition method.

A novel electrochemical strategy for NT-proBNP detection using IMFET for monitoring heart failure by saliva analysis.

Heart failure (HF) is a chronic cardiovascular disease that represents main cause of mortality worldwide, particularly for elderly. N-terminal pro-brain natriuretic peptide (NT-proBNP) was identified as the gold standard biomarker for HF diagnosis and therapy monitoring. Presently, saliva analysis represents an emerging and powerful tool for clinical applications and electrochemical immunosensors have shown their potential in Healthcare applications as selective and reliable systems for detecting clinical biomarkers. This work presents the detection of NT-proBNP in saliva samples by an immunologically modified Field effect Transistor (IMFET). TESUD ((11-triethoxysilyl) undecanal) was used as cross-linker to immobilise anti-NT-proBNP antibody onto the device. Our IMFET that was then tested in different matrices (e.g. phosphate buffered saline (PBS), artificial saliva and human saliva) using electrochemical impedance spectroscopy (EIS), and it resulted selective to NT-proBNP with good sensitivity (detection limit of 0.02 pg/mL) and a wide linear range (0.02-1 pg/mL and 0.5-20 pg/mL). Finally, NT-proBNP concentration in ten saliva samples was determined by performing the standard addition method. An enzyme-linked immunosorbent assay was used for confirming IMFET results, highlighting both IMFET accuracy (analyte recovery of 99 ± 8%) and precision (coefficient of variation always <10%), and supporting the suitability of the device for determining salivary NT-proBNP.

A readout circuit realizing electrochemical impedance spectroscopy for FET-based biosensors.

Electrochemical impedance spectroscopy (EIS) is a useful approach for modeling the equivalent circuit of biosensors such as field-effect transistor (FET)-based biosensors. During the process of sensor development, laboratory potentiostats are mainly used to realize the EIS. However, those devices are normally not applicable for real use-cases outside the laboratory, so miniaturized and optimized instrumentations are needed. Various integrated circuits (IC) are available that provide EIS, but these make developed systems highly dependent on semiconductor manufacturers, including component availability. In addition, these generally do not meet the instrumentation requirements for FET-based biosensors, thus external circuitry is necessary as well. In this work, an instrumentation is presented that performs EIS between 10 Hz and 100 kHz for FET-based biosensors. The instrumentation includes the generation of the excitation signal, the configuration of the semiconductor and the readout circuit. The readout circuit consists of a transimpedance amplifier with automatic gain adjustment, filter stages, a magnitude and a phase detection circuit. Since magnitude and phase are converted to a DC signal, digitization of the results is trivial without further signal processing steps, minimizing the computational load on the microcontroller. The transmission behavior of the magnitude and phase measurement circuits shows a high linearity for sinusoidal signals. Furthermore, the overall system was tested with resistors, whereby the magnitude measurement error (1.7%) and the phase shift error (1.6°) were determined within the working range of the instrumentation. The functionality of the instrumentation is demonstrated using pH-sensitive field-effect transistors (ISFET) in various solutions.Clinical relevance- Based on the electrochemical impedance spectroscopy of FET-based biosensors such as ImmunoFETs, new point-of-care testing (POCT) devices can be developed that e.g. quantitatively detect the concentration of biomarkers with very low detection limits in body fluids. The instrumentation presented in this work can be part of new generation of diagnostic tools featuring innovative sensor technologies.

A silicon nitride ISFET based immunosensor for tumor necrosis factor-alpha detection in saliva. A promising tool for heart failure monitoring.

According to the European statistics, approximately 26 million patients worldwide suffer from heart failure (HF), and this number seems to be steadily increasing. Inflammation plays a central role in the development of HF, and the pro-inflammatory cytokine Tumor necrosis factor-α (TNF-α) represents inflammation gold-standard biomarker. Early detection plays a crucial role for the prognosis and treatment of HF. An Ion Sensitive Field Effect Transistor (ISFET) based on silicon nitride transducer and biofunctionalized with anti-TNF-α antibody for label-free detection of salivary TNF-α is proposed. Electrochemical impedance spectroscopy (EIS) was used for TNF-α detection. Our ImmunoFET offered a detection limit of 1 pg mL-1, with an analytical reproducibility expressed by a coefficient of variance (CV) resulted < 10% for the analysis of saliva samples, and an analyte recovery of 94 ± 6%. In addition, it demonstrated high selectivity when compared to other HF biomarkers such as Inteleukin-10, N-terminal pro B-type natriuretic peptide, and Cortisol. Finally, ImmunoFET accuracy in determining the unknown concentration of TNF-α was successfully tested in saliva samples by performing standard addition method. The proposed ImmunoFET showed great promise as a complementary tool for biomedical application for HF monitoring by a non-invasive, rapid and accurate assessment of TNF-α.