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Accurate, robust, and rapid diagnostics is the basis for all well-functioning healthcare. There is a large need in point-of-care biosensors to facilitate diagnosis and reduce the need for cumbersome laboratory equipment. Proteases are key virulence factors in periodontitis. Periodontal disease is very common and characterized by inflammation and infection in the tooth-supporting structures and is linked to many systemic diseases such as cardiovascular disease, diabetes, and Alzheimers disease. Proteases present in periodontal disease, gingipains, are highly responsible for the disease onset and progression and are therefore a promising biomarker. Here we show a novel nanopore-based biosensor strategy for protease activity monitoring. Solid-state nanopores were modified with a proteolytic substrate, restricting the ionic current through the apertures of the nanopores. Protease can digest the proteolytic substrate thus enlarge the aperture and the ionic current. Trypsin was used as an initial model protease to investigate the performance of the sensor. We show that the solid-state nanoporebiosensor can detect trypsin with a limit of detection (LOD) of 0.005 ng/mL (0.2 pM). The detection system developed for the model enzyme was then applied to the detection of gingipains. The LOD for detection of gingipains was 1 ng/mL (0.02 nM), with a 27% recovery of the signal at 0.1 mu g/mL, indicating that the sensitivity and dynamic range are relevant for the clinical diagnosis of periodontitis. The generic detection of protease activity and high sensitivity make this a promising sensor technology for both diagnosis of periodontal disease and monitoring of other disease-related proteases. Funding Agencies|Swedish Strategic Research Foun-dation [SSF FFL15-0174, FFL15-0026]; Swedish Research Council [VR 2019-04690, VR 2017-04475, VR 2016-04874]; Swedish Cancer Foundation [CAN 2017/430]; Wallenberg Academy Fellow Program [KAW 2020.0190, KAW 2016.0231]

Matrix Metalloproteinase-7 (MMP-7) is a proteolytic enzyme overexpressed in different pathological conditions, including cancer, infection, and cardiovascular diseases, and is a relevant diagnostic biomarker and potential drug target. Here we demonstrate rapid and selective detection of MMP-7 with a limit-of-detection of 6 pg/mL and a dynamic range from 1 x 10(-2) to 1 x 10(3) ng/mL using a peptide decorated gold nanoparticle/carbon nanotube electrochemical sensor. The sensor could be operated in diluted human serum and synthetic urine, with high specificity towards MMP-7. Moreover, the integration of nanomaterials in the sensing electrode significantly increased the signal-to-background ratio and strongly improved the stability of the sensor when compared to a conventional gold electrode. The simple and cost-effective fabrication and the ease of use make this sensor a very promising protease detection device for diagnostics and drug development. Funding Agencies|Swedish Strategic Research FoundationSwedish Foundation for Strategic Research [SSF FFL15-0174, FFL15-0026]; Swedish Research CouncilSwedish Research Council [VR 2019-04690, VR 2017-04475]; Swedish Cancer Foundation [CAN 2017/430]; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University [2009-00971]; Wallenberg Academy Fellow Program [KAW 2015.0127, KAW 2016.0231]

WS2 nanotubes functionalized with carboxylic acid functions (WS2-COOH) were used for improved immobilization of the enzyme tyrosinase in order to form an electrochemical biosensor towards catechol and dopamine. The nanotubes were deposited on glassy carbon electrodes using a dispersion-filtration-transfer procedure to assure the reproducibility of the deposits. After the electrochemical and morphological characterization of these WS2-COOH nanotube deposits, the formed biosensors showed very satisfying performance towards catechol detection with a linear range of 0.6-70 μmol L-1 and a sensitivity of 10.7 ± 0.2 mA L mol-1. The apparent Michaelis Menten constant of this system is slightly lower than the KM value of tyrosinase in solution, reflecting an excellent accessibility of the active site of the enzyme combined with a good mass transport of the target molecule through the deposit. For dopamine detection, we observed an accumulation of this substrate due to electrostatic interactions between the amine function of dopamine and the carboxylic acid groups of the nanotubes. This led to improved signal capture at low dopamine concentrations. With linear ranges of 0.5-10 μmol L-1 and 10-40 μmol L-1, and respective sensitivities of 6.2 ± 0.7 mA L mol-1 and 3.4 ± 0.4 mA L mol-1, the overall sensor performance is within the average of comparable results using carbon nanotubes. Nonetheless, the simplified handling of these nanotubes and their reduced environmental impact make these WS2-COOH nanotubes a promising nanomaterial for biosensing applications.

A homemade gold electrode is modified with a carbon nanotubes/gold nanoparticles nanocomposite to perform selective and sensitive electrochemical detection of dengue toxin. This nanostructured composite offers a large specific surface and a reactive interface allowing the immobilization of biological material. Dengue antibodies are immobilized on gold nanoparticles via covalent bonding for dengue toxin detection. The porous tridimensional network of carbon nanotubes and gold nanoparticles enhances the electrochemical signal and the overall performance of the sensor. After optimization, the system exhibits a high sensitivity of − 0.44 ± 0.01 μA per decade with wide linear range between 1 × 10−12 and 1 × 10−6 g/mL at a working potential of 0.22 V vs Ag/AgCl. The extremely low detection limit (3 × 10−13 g/mL) ranks this immunosensor as one of the most efficient reported in the literature for the detection of recombinant viral dengue virus 2 NS1. This biosensor also offers good selectivity, characterized by a low response to various non-specific targets and assays in human serum. The outstanding performances and the reproducibility of the system place the biosensor developed among the best candidates for future medical applications and for early diagnosis of dengue fever. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00604-020-04339-y) contains supplementary material, which is available to authorized users.

A high performance impedimetric immunosensor for the dengue virus antibody detection is presented. The setup profits from the formation of controlled and reproducible carbon nanotube (CNT) deposits on electrodes. Their easy functionalization via electrogeneration of a polypyrrole-NHS (N-hydroxysuccinimido 11-(pyrrol-1-yl) undecanoate) film enables the immobilization of the Dengue Virus 2 NS1 glycoprotein, the receptor unit, on the porous CNT layer via covalent amide coupling to provide the necessary selectivity towards Dengue NS1 antibody. All building steps of this immunosensor and the performance of this system were monitored by impedance spectroscopy and cyclic voltammetry. The resulting impedimetric dengue biosensor was tested in bovine blood plasma in addition to conventional measurements under controlled environment. After optimization, this immunosensor shows a good linearity in a wide concentration range (10−13–10−5 g mL−1).

An original impedimetric immunosensor was developed based on carbon nanotube (CNT) deposits with controlled thicknesses for enhanced electroactive surface areas leading to improved sensor performances. Cholera monitoring was chosen as the model immune system for this setup. These CNT deposits were characterized using confocal laser microscopy and electrochemical methods. To form the sensor device, the CNT deposits were functionalized via electrocoating of polypyrrole-nitrilotriacetic acid (poly(pyrrole-NTA)) followed by the formation of a Cu (II) complex with the NTA functions. The bioreceptor unit, cholera toxin B Subunit, modified with biotin, was then immobilized via coordination of the biotin groups with the NTA-Cu(II) complex. Each step of the formation of the immunosensor and the subsequent binding of the analyte antibody anti-cholera toxin were investigated with cyclic voltammetry and Electrochemical Impedance Spectroscopy. After optimization, the resulting impedimetric cholera sensor shows excellent reproducibility, increased sensitivities, a very satisfying detection limit of 10−13 g mL−1 and an exceptional linear range for anti-cholera detection of 8 orders of magnitude (10−13–10−5 g mL−1) and a sensitivity of 24.7 ± 0.4 Ω per order of magnitude.

For four decades, the development of biointerfaces has been the subject of increasing research efforts in the field of biosensors and energy conversion. Owing to their high conductivity, inertness and electroactive surface area, graphene and carbon nanotube are widely used for the design of biosensors and their functionalization was successfully performed by electrogenerated polymers exhibiting affinity or covalent binding interactions towards biomolecules. Recent examples of electropolymerized films will be presented for the design of labeless immunosensors and aptasensors for bisphenol A, cocaine, dengue antibody or cholera toxin antibody [1]. We report thus the synthesis and electrochemical characterization of a novel electropolymerizable Ru(II) complex bearing an intercalator group as well as the electrochemical behavior and photoelectrochemical properties of the resulting polymer film [2]. The latter was applied to the label-free photoelectrochemical detection of cocaine. A photoelectrode was thus designed by electrodeposition of a pyrrole monomer modified with a polypyridyl Ru(II) complex bearing benzo[i]dipyrido-[3,2-a:2’.3’-c]phenazine (dppn) ligand. Owing to the intercalating properties of these immobilized complexes towards DNA double helix, cocaine aptamer was immobilized on the modified electrodes thanks to its stem-loop configuration in order to design a photoelectrochemical cocaine aptasensor. The detection of cocaine via the formation of the aptamer/cocaine complex was successfully observed via a decrease in the photocurrent intensity. The photoelectrochemical aptasensor exhibits a detection limit of 10 nmol L-1 and linear range of 1 10-8 to 5 10-4 mol L-1 [3]. We report here the original synthesis of an anti-bisphenol A (BPA) aptamer conjugated at its 5’-end with a pentahistidine peptide and its use for the design of an impedimetric BPA aptasensor. The introduction of a histidine tag is dedicated to the non-covalent binding of the aptamer onto electrodes previously modified by polypyrrole nitrilotriacetic films. The aptamer immobilization occurs via the histidine axial ligation onto Cu complex chelated by the polypyrrole film. This immobilization method, already used by our group to elaborate immunosensors or DNA sensors, offers a close proximity at the molecular level between the immobilized probe and the modified electrode surface. This approach thus confers a high sensitivity to the detection process, namely the aptamer/BPA folding. The resulting labeless impedimetric aptasensor displays a wide linear range from 10-11 to 10-6 mol L-1 with a sensitivity of 26 W cm2 per decade and an excellent specificity towards interfering agents such as 4,4'-dihydroxybiphenyl and bisphenol P. With the aim to improve the sensitivity of impedimetric biosensors, we have investigated the influence of a multiwalled carbon nanotube (MWCNT) coating and its thickness on the impedimetric transduction. We present thus a reliable procedure based on dispersion and filtration of MWCNT forming homogeneous films with controlled thicknesses and morphologies, which can easily be transferred on electrode surfaces. Polypyrrole-NTA films were electrogenerated on MWCNT modified glassy carbon electrode to immobilize biotinylated cholera toxin via the coordination of the biotin group onto Cu2+ complex chelated on the polymerized NTA groups. Concerning the design of this immunosensor, different thicknesses of carbon nanotube films were tested. For this purpose, three different volumes of CNT suspension solutions were filtered on a cellulose filter and then transferred to the electrode surface for modification of the three different thickness films by electropolymerization and post-biofunctionalization. The resulting impedimetric immunosensor for cholera toxin antibody, shows excellent reproducibility, increased sensitivities, and an exceptional linear range for antibody detection between 0.1 pg.mL-1 to 1 mg.mL-1. Similar results were obtained for the detection of Dengue antibody, namely a detection limit of 0.1 pg.mL-1. References F. Cecchini, L. Fajs, S. Cosnier, R. S. Marks, Trends in analytical chemistry, in press F. Haddache, A. Le Goff, B. Reuillard, K. Gorgy, C. Gondran, N. Spinelli, E. Defrancq, S. Cosnier, Chem. Eur. J. 2014, 20, 15555-15560. F. Haddache, A. Le Goff, N. Spinelli, P. Gairola, K. Gorgy, C. Gondran, E. Defrancq, S. Cosnier, Anal. Chem., submitted.