An Electroactive and Self-Assembling Bio-Ink, based on Protein-Stabilized Nanoclusters and Graphene, for the Manufacture of Fully Inkjet-Printed Paper-Based Analytical Devices.

Hundreds of new electrochemical sensors are reported in literature every year. However, only a few of them makes it to the market. Manufacturability, or rather the lack of it, is the parameter that dictates if new sensing technologies will remain forever in the laboratory in which they are conceived. Inkjet printing is a low-cost and versatile technique that can facilitate the transfer of nanomaterial-based sensors to the market. Herein, an electroactive and self-assembling inkjet-printable ink based on protein-nanomaterial composites and exfoliated graphene is reported. The consensus tetratricopeptide proteins (CTPRs), used to formulate this ink, are engineered to template and coordinate electroactive metallic nanoclusters (NCs), and to self-assemble upon drying, forming stable films. The authors demonstrate that, by incorporating graphene in the ink formulation, it is possible to dramatically improve the electrocatalytic properties of the ink, obtaining an efficient hybrid material for hydrogen peroxide (H2 O2 ) detection. Using this bio-ink, the authors manufactured disposable and environmentally sustainable electrochemical paper-based analytical devices (ePADs) to detect H2 O2 , outperforming commercial screen-printed platforms. Furthermore, it is demonstrated that oxidoreductase enzymes can be included in the formulation, to fully inkjet-print enzymatic amperometric biosensors ready to use.

Design of a photoelectrochemical lab-on-a-chip immunosensor based on enzymatic production of quantum dots in situ.

In this work we report the development and validation of a photoelectrochemical immunosensor on the basis of alkaline phosphatase (ALP)-linked immunoassay for the detection of human serum albumin as a model analyte. In this biosensor, oriented immobilization of capture antibodies on aminated polystyrene was achieved via physical adsorption. After the interaction with the analyte, ALP immobilised on the surface through the sandwich immunoassay catalyses the hydrolysis of sodium thiophosphate (TP) to hydrogen sulphide (H2S) which in the presence of cadmium ions yields CdS quantum dots (QDs). The electrical current is generated in the course of the photoelectrochemical process (PEC) during irradiation of the CdS QDs with a UV LED (365 nm) on home-made screen-printed carbon electrodes modified with a conductive polymer. Reaction time, steps and volumes were optimized for the miniaturization of the process in order to develop a lab-on-a-chip platform. The microfluidic system was designed with optimised parameters to fabricate the immunosensor combining the immunoassay with PEC detection. The final system presents a sensitivity comparable to that of the commercial kit thanks to the signal amplification enabled by the enzymatic growth of CdS QDs in situ. This photoelectrochemical immunosensing strategy potentially opens up a new avenue for the detection of a wide range of analytes of interest due to the universal and effective enzymatic signal amplification method. Moreover, the developed bioanalytical device allows for a great reduction of time and reagents compared to exiting commercial assays, making it suitable for point-of-care applications.

Development of portable CdS QDs screen-printed carbon electrode platform for electrochemiluminescence measurements and bioanalytical applications.

In this work, a portable and disposable screen-printed electrode-based platform for CdS QDs electrochemiluminescence (ECL) detection is presented. CdS QDs were synthesized in aqueous media and placed on top of carbon electrodes by drop casting. The CdS QDs spherical assemblies consisted of nanoparticles about 4 nm diameters and served as ECL sensitizers to enzymatic assays. The nanoparticles were characterized by optical techniques, TEM and XPS. Besides, the electrode modification process was optimized and further studied by SEM and confocal microscopy. The ECL emission from CdS QDs was triggered with H2O2 as cofactor and enzymatic assays were employed to modulate the CdS QDs ECL signal by blocking the surface or generating H2O2 in situ. Thiol-bearing compounds such as thiocholine generated through the hydrolysis of acetylthiocholine by acetylcholinesterase (AChE) interacted with the surface of CdS QDs thus blocking the ECL. The biosensor showed a linear range up to 5 mU mL-1 and a detection limit of 0.73 mU mL-1 for AChE. Moreover, the inhibition mechanism of the enzyme was studied by using 1,5-bis-(4-allyldimethylammonium-phenyl)pentan-3-one dibromide with a detection limit of 79.22 nM. Furthermore, the natural production of H2O2 from the oxidation of methanol by the action of alcohol oxidase was utilized to carry out the ECL process. This enzymatic assay presented a linear range up to 0.5 mg L-1 and a detection limit of 61.46 µg L-1 for methanol. The reported methodology shows potential applications for the development of sensitive and easy to hand biosensors and was applied to the determination of AChE and methanol in real samples.

SERS-based immunoassay for monitoring cortisol-related disorders.

As a natural response to a stressful situation, the human body produces cortisol. For this reason, cortisol is also called "the stress hormone" and is considered to be the principal stress biomarker. Although cortisol response to stress is essential for survival, abnormal levels in biological fluids may represent serious health risks. In this work, we present a cortisol biosensor which relies on a highly sensitive technique (surface-enhanced Raman spectroscopy, SERS) and a specific recognition (immunoassay). Gold nanostars were used as SERS nanotags, since they provided a better response than nanorods or nanospheres. Using the same concept, two different immunoassay modalities were evaluated, using either magnetic beads or gold-coated glass slides decorated with cortisol antibodies as the capture substrates. The magnetically-assisted SERS immunoassay presented a better performance and was therefore selected to quantify cortisol content in biological fluids (urine and serum). Significant advantages of this assay were found over standard methods such as Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and Enzyme-Linked Immunosorbent Assay (ELISA), including higher sensitivity and repeatability, minimum sample preparation, simplicity, and portability. Therefore, the proposed SERS immunoassay might be implemented as a highly efficient tool for in situ monitoring of human stress levels and cortisol-related disorders (e.g. Cushing's syndrome and Addison's disease).

Antibody-Directed Synthesis of Catalytic Nanoclusters for Bioanalytical Assays.

Synthesis of atomic nanoclusters (NCs) using proteins as a scaffold has attracted great attention. Usually, the synthetic conditions for the synthesis of NCs stabilized with proteins require extreme pH values or temperature. These harsh reaction conditions cause the denaturation of the proteins and end up in the loss of their biological functions. Until now, there are no examples of the use of antibodies as NC stabilizers. In this work, we present the first method for the synthesis of catalytic NCs that uses antibodies for the stabilization of NCs. Anti-BSA IgG was used as a model to demonstrate that it is possible to use an antibody as a scaffold for the synthesis of semiconductor and metallic NCs with catalytic properties. The synthesis of antibodies modified with NCs is carried out under nondenaturing conditions, which do not affect the antibody structure. The resulting antibodies still maintain the affinity for target antigens and protein G. The catalytic properties of the anti-BSA IgG modified with NCs can be used to the quantification of bovine serum albumin (BSA) in a direct sandwich enzyme-linked immunosorbent assay (ELISA).

Modification of chlorosulfonated polystyrene substrates for bioanalytical applications.

In this work the modification of polystyrene micro-well plates and their use as bioanalytical platform is described. A wet-chemical procedure was applied for the chlorosulfonation of these polystyrene substrates (PS) resulting in well-controlled and reactive surfaces. This method enabled the production of transparent and stable substrates under ambient conditions. The chlorosulfonyl moieties at the substrate surface were converted under mild conditions into different functional groups. The modification of PS served to increase the hydrophilic properties of the surface and thus, the improvement of interaction with biocompounds. The resulting substrates were characterized by contact angle measurements, X-ray Photoelectron Spectroscopy and colorimetry. PS substrates modified with different functional groups and attachment approaches (covalent link and direct adsorption of the antibodies) were used as the platform for immunoassays and the results compared to a commercial Human Serum Albumin ELISA kit. Aminated surfaces gave better results than those with carboxyl, alkene or epoxy groups and even the commercial kit.

Cellulose as an Inert Scaffold in Plasmon-Assisted Photoregeneration of Cofactor Molecules.

Plasmonic nanoparticles exhibit excellent light-harvesting properties in the visible spectral range, which makes them a convenient material for the conversion of light into useful chemical fuel. However, the need for using surface ligands to ensure colloidal stability of nanoparticles inhibits their photochemical performance due to the insulating molecular shell hindering the carrier transport. We show that cellulose fibers, abundant in chemical functional groups, can serve as a robust substrate for the immobilization of gold nanorods, thus also providing a facile way to remove the surfactant molecules. The resulting functional composite was implemented in a bioinspired photocatalytic process involving dehydrogenation of sodium formate and simultaneous photoregeneration of cofactor molecules (NADH, nicotinamide adenine dinucleotide) using visible light as an energy source. By systematic screening of experimental parameters, we compare photocatalytic and thermocatalytic properties of the composite and evaluate the role of palladium cocatalyst.

CdS quantum dots generated in-situ for fluorometric determination of thrombin activity.

A method is presented for sensitive determination of thrombin activity. It is based on (a) the interaction between fibrinogen after activation with thrombin, and (b) an enzymatic amplification step consisting of in-situ growth of CdS quantum dots (QDs). Fibrinogen is immobilized on the surface of the wells of a microplate and then incubated with a mixture of biotinylated fibrinogen and thrombin. Thrombin activates immobilized fibrinogen and free biotinylated fibrinogen. This leads to the formation of insoluble biotinylated fibrin that remains bound on the surface of the wells. Afterwards, the samples are incubated with avidin-labeled alkaline phosphatase (ALP) which binds to biotinylated fibrin. ALP hydrolyzes the substrate p-nitrophenyl phosphate (pNPP) under formation of phosphate ions which stabilize CdS QDs that are grown in-situ from cadmium(II) and sulfide. The generation of fibrin is correlated with the activity of thrombin. Increased thrombin concentration results in increased fluorescence that can be measured at excitation/emission wavelengths of 300/510 nm. The introduction of such an amplification step (the enzyme-triggered growth of QDs) allows for the quantification of thrombin in the picomolar concentration range, with a linear response up to 2.5 pM and a detection limit of 0.05 pM. The method was applied to the determination of thrombin activity in human plasma and of the thrombin inhibitor argatroban. Graphical abstract Schematic representation of a fluorometric method for determination of thrombin activity in the picomolar concentration range based on the interaction between fibrinogen after activation with thrombin, and an enzymatic amplification step consisting of in-situ growth of CdS quantum dots (CdS QD).

Biosensing strategies based on enzymatic reactions and nanoparticles.

Enzymes are pivotal elements in bioanalysis due to their specificity and extremely high catalytic activity. The sensitivity of bioanalytical assays depends mainly on the capacity of an observer to detect the product(s) of a biocatalytic reaction. Both natural and artificial compounds have been traditionally used to evaluate enzymatic activities. The drawbacks of chromogenic and fluorogenic organic enzymatic substrates are their high cost and low stability, resulting in high background signals. We review here state of the art assays in the detection of enzymatic activities using recent advances in nanoscience. Novel methods based on the use of nanoparticles lead to increased sensitivity and decreased costs for bioanalysis based on enzymes as recognition elements and signal amplifiers in Enzyme-Linked Immunosorbent Assays (ELISA). Novel approaches toward the detection of enzymatic activities are based on biocatalytic synthesis, modulation, etching, and aggregation of nanoparticles under physiological conditions.

Specific bioanalytical optical and photoelectrochemical assays for detection of methanol in alcoholic beverages.

Methanol is a poison which is frequently discovered in alcoholic beverages. Innovative methods to detect methanol in alcoholic beverages are being constantly developed. We report for the first time a new strategy for the detection of methanol using fluorescence spectroscopy and photoelectrochemical (PEC) analysis. The analytical system is based on the oxidation of cysteine (CSH) with hydrogen peroxide (H2O2) enzymatically generated by alcohol oxidase (AOx). H2O2 oxidizes capping agent CSH, modulating the growth of CSH-stabilized cadmium sulphide quantum dots (CdS QDs). Disposable screen-printed carbon electrodes (SPCEs) modified with a conductive osmium polymer (Os-PVP) complex were employed to quantify resulting CdS QDs. This polymer facilitates the "wiring" of in situ enzymatically generated CdS QDs, which photocatalyze oxidation of 1-thioglycerol (TG), generating photocurrent as the readout signal. Likewise, we proved that our systems did not suffer from interference by ethanol. The PEC assays showed better sensitivity than conventional methods, covering a wide range of potential applications for methanol quantification.

Photoelectrochemical detection of copper ions by modulating the growth of CdS quantum dots.

We discovered that copper ions (Cu2+) catalyze the oxidation of cysteine (CSH) by oxygen (O2) to modulate the growth of CSH-capped cadmium sulfide (CdS) nanoparticles (NPs). This new chemical process was applied to sensitive fluorogenic and photoelectrochemical (PEC) detection of Cu2+ ions in real samples of mineral and tap water using the photocatalytic activity of the resulting NPs. Disposable screen-printed electrodes (SPCEs) modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP) by cyclic voltammetry (CV) were employed for PEC analytical system. CdS NPs formed during the assay photocatalyze oxidation of 1-thioglycerol (TG) upon application of 0.3 V vs. Ag/AgCl to SPCEs. Os-PVP complex mediated the electron transfer between the electrode surface and CdS NPs. We proved that our assays did not suffer from interference from other ions accompanying Cu2+ and the sensitivity of our assays covers the European Union standard limit of Cu2+ ions in drinking water.

Microbead QD-ELISA: Microbead ELISA Using Biocatalytic Formation of Quantum Dots for Ultra High Sensitive Optical and Electrochemical Detection.

Electrochemical detection strategies employing semiconductor quantum dots (QDs) open up new opportunities for highly sensitive detection of biological targets. We designed a new assay based on microbead linked enzymatic generation of CdS QDs (Microbead QD-ELISA) and employed it in optical and electrochemical affinity assays for the cancer biomarker superoxide dismutase 2 (SOD2). Biotinylated antibodies against SOD2 were immobilized on the surface of polyvinyl chloride microbeads bearing streptavidin. In order to prevent any non-specific adsorption the microbeads were further blocked with bovine serum albumin. The analyte, SOD2 was captured on microbeads and labeled with alkaline phosphatase-conjugated antibody linked with mouse antibody against SOD2. Hydrolysis of para-nitrophenylphosphate by immobilized alkaline phosphatase triggered the rapid formation of phosphate-stabilized CdS QDs on the surface of microbeads. The resulting semiconductor nanoparticles were detected by fluorescence spectroscopy, microscopy, and square-wave voltammetry (SWV). The electrochemical assay based on the detection with square-wave voltammograms of Cd2+ ions originating from immobilized CdS QDs showed linearity up to 45 ng mL-1, and the limit of SOD2 detection equal to 0.44 ng mL-1 (1.96 × 10-11 M). This detection limit is lower by 2 orders of magnitude in comparison with that of other previously published assays for superoxide dismutase. The electrochemical assay was validated with HepG2 (Human hepatocellular carcinoma) cell lysate containing SOD2.

Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors.

The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV-vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine.

Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP).

Application of photocatalytic cadmium sulfide nanoparticles to detection of enzymatic activities of glucose oxidase and glutathione reductase using oxidation of 3,3',5,5'-tetramethylbenzidine.

It was found out that semiconductor CdS nanoparticles (NPs) are able to catalyze photooxidation of the well known chromogenic enzymatic substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen. The photocatalytical oxidation of TMB does not require hydrogen peroxide and its rate is directly proportional to the quantity of CdS NPs produced in situ through the interaction of Cd(2+) and S(2-) ions in an aqueous medium. This phenomenon was applied to development of colorimetric sensitive assays for glucose oxidase and glutathione reductase based on enzymatic generation of CdS NPs acting as light-powered catalysts. Sensitivity of the developed chromogenic assays was of the same order of magnitude or even better than that of relevant fluorogenic assays. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for a number of enzymes using inexpensive and available TMB as a universal chromogenic compound.

Peroxidase-mimicking DNAzyme modulated growth of CdS nanocrystalline structures in situ through redox reaction: application to development of genosensors and aptasensors.

This work demonstrates the use of the peroxidase-mimicking DNAzyme (peroxidase-DNAzyme) as general and inexpensive platform for development of fluorogenic assays that do not require organic fluorophores. The system is based on the affinity interaction between the peroxidase-DNAzyme bearing hairpin sequence and the analyte (DNA or low molecular weight molecule), which changes the folding of the hairpin structure and consequently the activity of peroxidase-DNAzyme. Hence, in the presence of the analyte the peroxidase-DNAzyme structure is disrupted and does not catalyze the aerobic oxidation of l-cysteine to cystine. Thus, l-cysteine is not removed from the system and the fluorescence of the assay increases due to the in situ formation of fluorescent CdS nanocrystals. The capability of the system as a platform for fluorogenic assays was demonstrated through designing model geno- and aptasensor for the detection of a tumor marker DNA and a low molecular weight analyte, adenosine 5'triphosphate (ATP), respectively.

Enzymatic etching of gold nanorods by horseradish peroxidase and application to blood glucose detection.

Gold nanorods (AuNRs) have become some of the most used nanostructures for biosensing and imaging applications due to their plasmon-related optical response, which is highly sensitive toward minute changes in the AuNR aspect ratio. In this context, H2O2 has been used to trigger the chemical etching of AuNRs, thereby inducing a decrease of their aspect ratio. However, special conditions and relatively high concentrations of H2O2 are usually required, preventing the applicability of the system for biodetection purposes. To overcome this limitation we have introduced a biocatalytic species, the enzyme horseradish peroxidase (HRP) that is able to induce a gradual oxidation of AuNRs in the presence of trace concentrations of H2O2. Interestingly, the presence of halide ions has also been found to be essential for this process. As a consequence, other enzymatic reactions, such as those catalyzed by glucose oxidase, can be easily coupled to HRP activity, allowing the detection of different amounts of glucose. On the basis of these findings, we developed a highly sensitive and simple colorimetric assay that can be read out by the naked eye and allows the detection of physiological glucose concentrations in human serum.

Thiocholine mediated stabilization of in situ produced CdS quantum dots: application for the detection of acetylcholinesterase activity and inhibitors.

The use of acetylcholinesterase (AChE) inhibitors as chemical warfare agents or pesticides represents a strong hazard against human health. The high toxicity of these compounds arises from their ability to inhibit acetylcholinesterase from degrading acetylcholine (ACh), which could affect the physiology of the nervous system with serious or fatal consequences. Here we report a simple and fluorimetric system for a highly sensitive detection of AChE activity and inhibitors. The principle of this approach is based on the hydrolysis of acetylthiocholine (ATCh) by AChE, which yields the thiol-bearing compound thiocholine (TCh) that at trace concentrations stabilized the in situ generated CdS quantum dots (QDs). The system shows a linear relationship between the fluorescence intensity and AChE activity from 1 to 10 mU mL(-1) in buffer solution. The accuracy of the proposed system was further demonstrated through the determination of AChE activity in human serum (HS) by the standard addition method. Furthermore, this novel and highly sensitive sensing system allows the detection of 80 pM of the AChE inhibitor paraoxon and 100 nM of galanthamine. The reported methodology shows potential applications for the development of a simple and inexpensive assay for the routine quantification of AChE activity and inhibitors.

Aminoferrocene-based prodrugs and their effects on human normal and cancer cells as well as bacterial cells.

Aminoferrocene-based prodrugs are activated under cancer-specific conditions (high concentration of reactive oxygen species, ROS) with the formation of glutathione scavengers (p-quinone methide) and ROS-generating iron complexes. Herein, we explored three structural modifications of these prodrugs in an attempt to improve their properties: (a) the attachment of a -COOH function to the ferrocene fragment leads to the improvement of water solubility and reactivity in vitro but also decreases cell-membrane permeability and biological activity, (b) the alkylation of the N-benzyl residue does not show any significant affect, and (c) the attachment of the second arylboronic acid fragment improves the toxicity (IC50) of the prodrugs toward human promyelocytic leukemia cells (HL-60) from 52 to 12 µM. Finally, we demonstrated that the prodrugs are active against primary chronic lymphocytic leukemia (CLL) cells, with the best compounds exhibiting an IC50 value of 1.5 µM. The most active compounds were found to not affect mononuclear cells and representative bacterial cells.

Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots.

In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) leads to the formation of p-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd(2+) with S(2-) ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL(-1) of analyte antibody with a linear range up to 10 ng mL(-1). The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric p-nitrophenyl phosphate assay.