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.

Fast impedimetric immunosensing of IgGs associated with peanut and hazelnut allergens.

Food allergies trigger a variety of clinical adverse symptoms and clinical evidence suggests that the presence of food allergy-related IgG can be helpful in the diagnosis when analyzed at the peptide-epitope level. To validate and select the peptides based on their specificity toward hazelnut or peanut epitopes, the authors of this study developed a silicon-based microchip coupled with click-chemistry bound peptides identified by the Fraunhofer Institute for Cell Therapy and Immunology. Peptides related to hazelnut and peanut allergies were identified and used to develop a silicon-based microchip. Peptides were coupled with click-chemistry to the sensor surface. The immunosensor was developed by electrografting diazotized amino phenylacetic acid and subsequently, dibenzocyclooctyne-amine (DBCO-NH2) was used as click-chemistry to allow coupling of the peptides with a C-terminal linker and azide structure. Energy-dispersive X-ray spectroscopy, electrochemical impedance spectroscopy (EIS), and fluorescence microscopy techniques have been used to analyze the bio-functionalization of the developed electrode. The peptide-epitope recognition was studied for seven allergen-derived peptides. The electrochemical responses were studied with sera from rabbits immunized with hazelnut and peanut powder. The microchips functionalized with the chosen peptides (peanut peptides T12 and EO13 and hazelnut peptides S4 and EO14 with an RSD of 4%, 3%, 9%, and 1% respectively) demonstrated their ability to specifically detect prevalent anti-nut related IgGs in rabbit sera in a range of dilutions from 1:500000 (0.0002%) until 1:50000 (0.002%). In addition, the other peptides showed promising differentiation abilities which can be further studied to perform multivariable detection fingerprint of anti-allergens in blood sera.

A Sensitive Micro Conductometric Ethanol Sensor Based on an Alcohol Dehydrogenase-Gold Nanoparticle Chitosan Composite.

In this paper, a microconductometric sensor has been designed, based on a chitosan composite including alcohol dehydrogenase-and its cofactor-and gold nanoparticles, and was calibrated by differential measurements in the headspace of aqueous solutions of ethanol. The role of gold nanoparticles (GNPs) was crucial in improving the analytical performance of the ethanol sensor in terms of response time, sensitivity, selectivity, and reproducibility. The response time was reduced to 10 s, compared to 21 s without GNPs. The sensitivity was 416 µS/cm (v/v%)-1 which is 11.3 times higher than without GNPs. The selectivity factor versus methanol was 8.3, three times higher than without GNPs. The relative standard deviation (RSD) obtained with the same sensor was 2%, whereas it was found to be 12% without GNPs. When the air from the operator's mouth was analyzed just after rinsing with an antiseptic mouthwash, the ethanol content was very high (3.5 v/v%). The background level was reached only after rinsing with water.

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.

One-step impedimetric NT-proBNP aptasensor targeting cardiac insufficiency in artificial saliva.

Currently, sensitive and accurate approaches for diagnosis, rapid assessment, and cardiac biomarker monitoring in patients with heart failure are needed. In this context, the advantages of aptamers over traditional antibodies have been employed to fabricate a single-step impedimetric N-terminal pro b-type natriuretic peptide (NT-proBNP)-modified gold microelectrode array. The development of an electrochemical aptasensing platform was based on the coimmobilization of alkanethiol self-assembled monolayers and amine-terminated aptamer that specifically recognized cardiac NT-proBNP protein resulting in charge electron transfer. Electroimpedimetric signals of the sensor were observed to be linear to the NT-proBNP concentrations in the range of 5.0 × 10-3 to 1.0 pg mL-1 (R2 = 0.9624), while achieving a low detection limit of 5.0 × 10-3 pg mL-1. Clinically relevant detection levels for NT-proBNP were achieved in a simple, rapid, and label-free measurement using artificial saliva, which was highlighted to be specific, regenerative, and selective over potential interferers occurring during the processes of cardiac insufficiency, Therefore, the novel NT-proBNP aptasensor is a promising point-of-care tool exhibiting safe, non-invasive, affordable, and non-prescription home use accessible to overcome the limitations associated with conventional ELISA and previous aptasensing.

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 Novel Cortisol Immunosensor Based on a Hafnium Oxide/Silicon Structure for Heart Failure Diagnosis.

Assessing cortisol levels in human bodies has become essential to diagnose heart failure (HF). In this work, we propose a salivary cortisol detection strategy as part of an easily integrable lab-on-a-chip for detection of HF biomarkers. Our developed capacitive immunosensor based on hafnium oxide (HfO2)/silicon structure showed good linearity between increasing cortisol concentration and the charge-transfer resistance/capacitance. Moreover, the developed biosensor was demonstrated to be highly selective toward cortisol compared to other HF biomarkers such as tumor necrosis factor (TNF-α) and N-terminal pro-brain natriuretic peptide (NT-proBNP). The precision of our developed biosensor was evaluated, and the difference between the determined cortisol concentration in saliva and its expected one is <18%.

Selective Antibody-Free Sensing Membranes for Picogram Tetracycline Detection.

As an antibody-free sensing membrane for the detection of the antibiotic tetracycline (TC), a liquid PVC membrane doped with the ion-pair tetracycline/θ-shaped anion [3,3'-Co(1,2-C2B9H11)2]- ([o-COSAN]-) was formulated and deposited on a SWCNT modified gold microelectrode. The chosen transduction technique was electrochemical impedance spectroscopy (EIS). The PVC membrane was composed of: the tetracycline/[o-COSAN]- ion-pair, a plasticizer. A detection limit of 0.3 pg/L was obtained with this membrane, using bis(2-ethylhexyl) sebacate as a plasticizer. The sensitivity of detection of tetracycline was five times higher than that of oxytetracycline and of terramycin, and 22 times higher than that of demeclocycline. A shelf-life of the prepared sensor was more than six months and was used for detection in spiked honey samples. These results open the way to having continuous monitoring sensors with a high detection capacity, are easy to clean, avoid the use of antibodies, and produce a direct measurement.

Electrochemical Impedance Spectroscopy Microsensor Based on Molecularly Imprinted Chitosan Film Grafted on a 4-Aminophenylacetic Acid (CMA) Modified Gold Electrode, for the Sensitive Detection of Glyphosate.

A novel electrochemical impedance spectroscopy (EIS) microsensor was implemented for the dosage of traces of glyphosate, in real and synthetic water samples. Molecularly imprinted chitosan was covalently immobilized on the surface of the microelectrode previously modified with 4-aminophenylacetic acid (CMA). The characterization of the resulting microelectrodes was carried out by using cyclic voltammetry measurement (CV), scanning electron microscopy (SEM), and electrochemical impedance spectrometry (EIS). EIS responses of the CS-MIPs/CMA/Au microsensor toward GLY was well-proportional to the concentration in the range from 0.31 × 10-9 to 50 × 10-6 mg/mL indicating a good correlation. The detection limit of GLY was 1 fg/mL (S/N = 3). Moreover, this microsensor showed good reproducibility and repeatability, high selectivity, and can be used for the detection of GLY in river water.

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.

Directed Self-Assembly of Block Copolymers for the Fabrication of Functional Devices.

Directed self-assembly of block copolymers is a bottom-up approach to nanofabrication that has attracted high interest in recent years due to its inherent simplicity, high throughput, low cost and potential for sub-10 nm resolution. In this paper, we review the main principles of directed self-assembly of block copolymers and give a brief overview of some of the most extended applications. We present a novel fabrication route based on the introduction of directed self-assembly of block copolymers as a patterning option for the fabrication of nanoelectromechanical systems. As a proof of concept, we demonstrate the fabrication of suspended silicon membranes clamped by dense arrays of single-crystal silicon nanowires of sub-10 nm diameter. Resulting devices can be further developed for building up high-sensitive mass sensors based on nanomechanical resonators.

Exploring Strategies to Contact 3D Nano-Pillars.

This contribution explores different strategies to electrically contact vertical pillars with diameters less than 100 nm. Two process strategies have been defined, the first based on Atomic Force Microscope (AFM) indentation and the second based on planarization and reactive ion etching (RIE). We have demonstrated that both proposals provide suitable contacts. The results help to conclude that the most feasible strategy to be implementable is the one using planarization and reactive ion etching since it is more suitable for parallel and/or high-volume manufacturing processing.

Development of Novel Magneto-Biosensor for Sulfapyridine Detection.

In this work, we report the development of a highly sensitive biosensor for sulfapyridine detection based on an integrated bio micro-electromechanical system (Bio-MEMS) containing four gold working electrodes (WEs), a platinum counter electrode (CE), and a reference electrode (RE). Firstly, the cleaned WEs were modified with 4-aminophenylacetic acid (CMA). Then, (5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2BSA) was immobilized onto the transducers surface by carbodiimide chemistry. The analyte was quantified by competitive detection with SA2BSA immobilized on the WE toward a mixture of Ab155 antibody (with fixed concentration) and sulfapyridine. In order to obtain a highly sensitive biosensor, Ab155 was immobilized onto magnetic latex nanoparticles surface to create a 3D architecture (Ab-MLNp). Using electrochemical impedance spectroscopy (EIS), we investigated the influence of the Ab-MLNp on the sensitivity of our approach. The optimized system was analyzed, as competitive assay, with different concentrations of sulfapyridine (40 µM, 4 µM, and 2 nM) and with phosphate buffer solution. From data fitting calculations and graphs, it was observed that the EIS showed more linearity when Ab-MLNp was used. This result indicates that the magnetic latex nanoparticles increased the sensitivity of the biosensor.

Capacitance electrochemical biosensor based on silicon nitride transducer for TNF-α cytokine detection in artificial human saliva: Heart failure (HF).

In the present study, we have developed a capacitance electrochemical biosensor based on silicon nitride substrate (Si3N4/SiO2/Si[P]/Al) for Tumour Necrosis Factor Alpha (TNF-α) cytokines detection. Micro-contact printing, Fluorescence microscopy characterization and contact angle measurement (CAM) were carried out during the bio-functionalization of the biosensor surface. Mott-Schottky analyses were applied for TNF-α detection within the range of 1 pg/mL to 30 pg/mL in which the immunosensor has exhibited a good linearity, a sensitivity of 4 mV.pM-1 and 4.4 mV.pM-1 in PBS and artificial saliva (AS) respectively. While the LOD was found at 0.38 pg/mL and 1 pg/mL in PBS and AS respectively. The developed immunosensor has also demonstrated a high and good selectivity for TNF-α detection in human AS when compared to other interferences like Cortisol and Interleukin-10. The performances of the developed biosensor are very promising for biomedical application to predict the first sign of inflammation.

Development and application of a novel electrochemical immunosensor for tetracycline screening in honey using a fully integrated electrochemical Bio-MEMS.

Tetracycline (TC) is a veterinary drug, wildly prescribed for prophylactic and therapeutic purposes. Consequently, its remaining residues in food products have to be regularized. We report in this paper about the development of a novel immunosensor based on an integrated bio micro-electromechanical system (Bio-MEMS) containing eight gold microelectrodes (µWEs), an integrated silver and platinum reference and counter electrodes, respectively. TC immobilization on the µWEs surface was conducted using three methods. The first through functionalization with 4-aminophenylacetic acid (CMA), the second by functionalization with CMA followed by preconcentration of a new structure of magnetic nanoparticles (MNPs) coated with poly (pyrrole-co-pyrrole-2-carboxylic acid) (Py/Py-COOH/MNPs) cross-linked with Ab-TC, and the last one directly through the functionalization with Py/Py-COOH/MNPs. The analyte was quantified by competitive detection with TC immobilized on the µWEs surface toward specific polyclonal antibody (Ab-TC), using a mixture of a fixed concentration of Ab-TC and decreasing levels of TC one from 0.1 pg mL-1 to 1000 pg mL-1. Microcontact printing, followed by fluorescence microscopy characterization were performed during the functionalization of the immunosensor surface to certify that the corresponding immune detection process is taking place. This immunosensor was found to be highly sensitive with a limit of detection of 1.2 pg mL-1 and specific in the presence of interferents. The standard addition method was exploited to detect TC in honey samples. The present immunosensor platform is up-and-coming for TC detection which can dramatically decrease the time of analysis providing a new pathway for advanced immunoassays development in industrial food control.

Biopatterning of antibodies on poly(pyrrole)-nanowires using nanocontact printing: Surface characterization.

A highly performant patterning of antibodies using poly(pyrrole) nanowires (PPy-NWs) was devised on thermoplastic surfaces based on silane derivatives. The PPy-NWs were fabricated employing nanocontact printing and controlled chemical polymerization (nCP-CCP) on poly(ethylene terephthalate), cyclic olefin copolymer, poly(ethylene 2,6-naphthalate), and polyimide. The technique used a commercial compact disk as a template (mold) to produce nanopatterned polydimethylsiloxane stamps. The nanopatterned stamp was then employed to print PPy-NWs. The printing technique permits to control PPy-NW size and shape. The dimensions of the printed PPy-NWs were: 785 ±â€¯1.5 nm (width), 174 ±â€¯2.1 nm (height), and a separation between wires of 540 ±â€¯1.2 nm. The printing process and the surface properties of the PPy-NWs pattern were successfully characterized by scanning electron microscopy and atomic force microscopy. Biopatterning was completed by the chemical immobilization of the specific anti-human interleukin-10 monoclonal antibody on PPy-NW using gluteraldehyde. The biocomposite was tested using qualitative immunocytokine bioassay, which is of great importance for early stage cancer detection. For that purpose, fluorescent imaging was used to certify the immunodetection of the recombinant human interleukin-10. The biopatterning technology provides a simple, low cost and one step procedure. Undoubtedly, this new technology will impact and provide an alternative to the current techniques applied for bioengineering and nanopatterning.

A low-cost and miniaturized potentiostat for sensing of biomolecular species such as TNF-α by electrochemical impedance spectroscopy.

Miniaturizing potentiostats, keeping their cost low and yet preserving full measurement characteristics (e.g. bandwidth, determination of capacitive/inductive contribution to sensor's impedance and parallel screening) is still an unresolved challenge in bioelectronics. In this work, the combination of simple analogue circuitry together with powerful microcontrollers and a digital filter implementation is presented as an alternative to complex and incomplete architectures reported in the literature. A low-cost acquisition electronic system fully integrated with a biosensors platform containing eight gold working microelectrodes and integrated reference and counter electrodes was developed and validated. The manufacturing cost of the prototype was kept below 300 USD. The performance of the proposed device was benchmarked against a commercial impedance analyzer through the electrochemical analysis of a highly sensitive biosensor for the detection of tumor necrosis factor α (TNF-α) within the randomly chosen range of 266pg/mL to 666ng/mL in physiological medium (PBS). A strong correlation between the outputs of both devices was found in a critical range of frequencies (1-10Hz), and several TNF-α cytokine concentrations were properly discriminated. These results are very promising for the development of low-cost, portable and miniaturized electrochemical systems for point-of-care and environmental diagnosis.

A fully integrated electrochemical biosensor platform fabrication process for cytokines detection.

Interleukin-1b (IL-1b) and interleukin-10 (IL-10) biomarkers are one of many antigens that are secreted in acute stages of inflammation after left ventricle assisted device (LVAD) implantation for patients suffering from heart failure (HF). In the present study, we have developed a fully integrated electrochemical biosensor platform for cytokine detection at minute concentrations. Using eight gold working microelectrodes (WEs) the design will increase the sensitivity of detection, decrease the time of measurements, and allow a simultaneous detection of varying cytokine biomarkers. The biosensor platform was fabricated onto silicon substrates using silicon technology. Monoclonal antibodies (mAb) of anti-human IL-1b and anti-human IL-10 were electroaddressed onto the gold WEs through functionalization with 4-carboxymethyl aryl diazonium (CMA). Cyclic voltammetry (CV) was applied during the WE functionalization process to characterize the gold WE surface properties. Finally, electrochemical impedance spectroscopy (EIS) characterized the modified gold WE. The biosensor platform was highly sensitive to the corresponding cytokines and no interference with other cytokines was observed. Both cytokines: IL-10 and IL-1b were detected within the range of 1pgmL-1 to 15pgmL-1. The present electrochemical biosensor platform is very promising for multi-detection of biomolecules which can dramatically decrease the time of analysis. This can provide data to clinicians and doctors concerning cytokines secretion at minute concentrations and the prediction of the first signs of inflammation after LVAD implantation.

Novel strategy for sulfapyridine detection using a fully integrated electrochemical Bio-MEMS: Application to honey analysis.

Sulfapyridine (SPy) is a sulfonamide antibiotic largely employed as veterinary drugs for prophylactic and therapeutic purposes. Therefore, its spread in the food products has to be restricted. Herein, we report the synthesis and characterization of a novel electrochemical biosensor based on gold microelectrodes modified with a new structure of magnetic nanoparticles (MNPs) coated with poly(pyrrole-co-pyrrole-2-carboxylic acid) (Py/Py-COOH) for high efficient detection of SPy. This analyte was quantified through a competitive detection procedure with 5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid-BSA (SA2-BSA) antigens toward polyclonal antibody (Ab-155). Initially, gold working electrodes (WEs) of integrated biomicro electro-mechanical system (BioMEMS) were functionalized by Ppy-COOH/MNPs, using a chronoamperometric (CA) electrodeposition. Afterward, SA2-BSA was covalently bonded to Py/Py-COOH/MNP modified gold WEs through amide bonding. The competitive detection of the analyte was made by a mixture of a fixed concentration of Ab-155 and decreasing concentrations of SPy from 50µgL-1 to 2ngL-1. Atomic Force Microscopy characterization was performed in order to ensure Ppy-COOH/MNPs electrodeposition on the microelectrode surfaces. Electrochemical measurements of SPy detection were carried out using electrochemical impedance spectroscopy (EIS). This biosensor was found to be highly sensitive and specific for SPy, with a limit of detection of 0.4ngL-1. This technique was exploited to detect SPy in honey samples by using the standard addition method. The measurements were highly reproducible for detection and interferences namely, sulfadiazine (SDz), sulfathiazole (STz) and sulfamerazine (SMz). Taking these advantages of sensitivity, specificity, and low cost, our system provides a new horizon for development of advanced immunoassays in industrial food control.

Nanoparticles with tunable shape and composition fabricated by nanoimprint lithography.

Cone-like and empty cup-shaped nanoparticles of noble metals have been demonstrated to provide extraordinary optical properties for use as optical nanoanntenas or nanoresonators. However, their large-scale production is difficult via standard nanofabrication methods. We present a fabrication approach to achieve arrays of nanoparticles with tunable shape and composition by a combination of nanoimprint lithography, hard-mask definition and various forms of metal deposition. In particular, we have obtained arrays of empty cup-shaped Au nanoparticles showing an optical response with distinguishable features associated with the excitations of localized surface plasmons. Finally, this route avoids the most common drawbacks found in the fabrication of nanoparticles by conventional top-down methods, such as aspect ratio limitation, blurring, and low throughput, and it can be used to fabricate nanoparticles with heterogeneous composition.