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.

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.

Dual Enzymatic Detection by Bulk Electrogenerated Chemiluminescence.

The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here, we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (ß-nicotinamide adenine dinucleotide (NADH) and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3](2+) at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths.

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.

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.

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.