Alfredo de la Escosura-Muñiz

Immunosensing using nanoparticles

Immunosensing technology is taking advantage of the lastest developments in materials science and inparticular from the nanomaterials field. Because of their unprecedented optical tunability as well as electrical and electrochemical qualities, we are seeing significant developments in the design of novel immunoassays; various conventional optical and electrical platforms which allow for future applications in several fields are being used. Properties of nanoparticles such as light absorption and dispersion are bringing interesting immunosensing alternatives. Nanoparticles are improving the sensitivity of existing techniques used for protein detection in immunoassays based on Surface Plasmon Resonance, Quartz Crystal Microbalance, Fluorescence spectroscopy etc. Electrochemical techniques are also taking advantage of electrical properties of nanoparticles. Redox properties of metal based nanoparticles, surface impedance change and conductance changes once nanoparticles are present as labelling tags or modifiers of transducer surfaces are also improving the technology. In most of the examples nanoparticle based biosensing systems are being offered as excellent screening and superior alternatives to existing conventional strategies/assays with interest for fields in clinical analysis, food quality, safety and security.

A nanochannel/nanoparticle-based filtering and sensing platform for direct detection of a cancer biomarker in blood.

A rapid nanochannel-based immunoassay capable of the filtering and subsequent detection of proteins in whole blood without any sample preparation is described. This is accomplished by using a nanoporous/nanochannel membrane modified with antibodies, the conductivity of which toward a redox indicator is tuned by primary and secondary immunoreactions with proteins and gold nanoparticles. This interesting nanopore blockage by gold nanoparticles is enhanced by silver deposition that further decreases the diffusion of the signaling indicator through the nanochannel. The efficiency of the nanochannels to act as immunoreaction platforms including the use of nanoparticles is also monitored by microscopic techniques. Successful detection of immunoglobulins including a cancer biomarker is achieved in buffer as well as in whole blood. This system constitutes an efficient immunoassay capable of detecting up to 52 U mL(-1) of CA15-3. The developed nanochannel/nanoparticle-based device can be used for several other proteins and extended also to DNA detection with interest not only for diagnostics but also environmental monitoring, food analysis, safety, and security applications.

Rapid identification and quantification of tumor cells using an electrocatalytic method based on gold nanoparticles.

There is a high demand for simple, rapid, efficient, and user-friendly alternative methods for the detection of cells in general and, in particular, for the detection of cancer cells. A biosensor able to detect cells would be an all-in-one dream device for such applications. The successful integration of nanoparticles into cell detection assays could allow for the development of this novel class of cell sensors. Indeed, their application could well have a great future in diagnostics, as well as other fields. As an example of a novel biosensor, we report here an electrocatalytic device for the specific identification of tumor cells that quantifies gold nanoparticles (AuNPs) coupled with an electrotransducing platform/sensor. Proliferation and adherence of tumor cells are achieved on the electrotransducer/detector, which consists of a mass-produced screen-printed carbon electrode (SPCE). In situ identification/quantification of tumor cells is achieved with a detection limit of 4000 cells per 700 microL of suspension. This novel and selective cell-sensing device is based on the reaction of cell surface proteins with specific antibodies conjugated with AuNPs. Final detection requires only a couple of minutes, taking advantage of the catalytic properties of AuNPs on hydrogen evolution. The proposed detection method does not require the chemical agents used in most existing assays for the detection of AuNPs. It allows for the miniaturization of the system and is much cheaper than other expensive and sophisticated methods used for tumor cell detection. We envisage that this device could operate in a simple way as an immunosensor or DNA sensor. Moreover, it could be used, even by inexperienced staff, for the detection of protein molecules or DNA strands.

Label-Free Impedimetric Aptasensor for Ochratoxin-A Detection Using Iridium Oxide Nanoparticles

In this article, a novel aptasensor for ochratoxin A (OTA) detection based on a screen-printed carbon electrode (SPCE) modified with polythionine (PTH) and iridium oxide nanoparticles (IrO2 NPs) is presented. The electrotransducer surface is modified with an electropolymerized film of PTH followed by the assembly of IrO2 NPs on which the aminated aptamer selective to OTA is exchanged with the citrate ions surrounding IrO2 NPs via electrostatic interactions with the same surface. Electrochemical impedance spectroscopy (EIS) in the presence of the [Fe(CN)6](-3/-4) redox probe is employed to characterize each step in the aptasensor assay and also for label-free detection of OTA in a range between 0.01 and 100 nM, obtaining one of the lowest limits of detection reported so far for label-free impedimetric detection of OTA (14 pM; 5.65 ng/kg). The reported system also exhibits a high reproducibility, a good performance with a white wine sample, and an excellent specificity against another toxin present in such sample.

Simple monitoring of cancer cells using nanoparticles.

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.

Controlling the electrochemical deposition of silver onto gold nanoparticles: Reducing interferences and increasing the sensitivity of magnetoimmuno assays

An electrocatalytical method induced by gold nanoparticles in order to improve the sensitivity of the magnetoimmunosensing technology is reported. Microparamagnetic beads as primary antibodies immobilization platforms and gold nanoparticles modified with secondary antibodies as high sensitive electrocatalytical labels are used. A built-in magnet carbon electrode allows the collection/immobilization on its surface of the microparamagnetic beads with the immunological sandwich and gold nanoparticle catalysts attached onto. The developed magnetoimmunosensing technology allows the antigen detection with an enhanced sensitivity due to the catalytic effect of gold nanoparticles on the electroreduction of silver ions. The main parameters that affect the different steps of the developed assay are optimized so as to reach a high sensitive electrochemical detection of the protein. The low levels of gold nanoparticles detected with this method allow the obtaining of a novel immunosensor with low protein detection limits (up to 23 fg/mL), with special interest for further applications in clinical analysis, food quality and safety as well as other industrial applications.

Gold nanoparticle-based electrochemical magnetoimmunosensor for rapid detection of anti-hepatitis B virus antibodies in human serum

A sandwich immunoassay using magnetic beads as bioreaction platforms and AuNPs as electroactive labels for the electrochemical detection of human IgG antibodies anti-Hepatitis B surface antigen (HBsAg), is here presented as an alternative to the standard methods used in hospitals for the detection of human antibodies directed against HBsAg (such as ELISA or MEIA). The electrochemical detection of AuNPs is carried out approaching their catalytic properties towards the hydrogen evolution in an acidic medium, without previous nanoparticle dissolution. The obtained results are a good promise toward the development of a fully integrated biosensing set-up. The developed technology based on this detection mode would be simple to use, low cost and integrated into a portable instrumentation that may allow its application even at doctor-office. The sample volumes required can be lower than those used in the traditional methods. This may lead to several other applications with interest for clinical control.

Nanochannels for diagnostic of thrombin-related diseases in human blood

A high sensitive voltammetric method for rapid determination of thrombin spiked in whole blood by taking advantage of both aptamer-based recognition and the use of a nanoporous membrane has been developed. The nanoporous membrane not only acts as platform for the thrombin recognition but also as filter of the micrometric components such as white and red blood cells, consequently minimizing matrix effects. The protocol involves a sandwich format in the inner walls (200 nm diameter) of an anodized alumina oxide filter membrane (AAO). The analytical signal, by DPV oxidation of [Fe(CN)(6)](4-), is based on the blockage in the pores which affects the diffusion of [Fe(CN)(6)](4-) to the screen-printed carbon electrotransducer (SPCEs) modified with the membrane. By labeling the anti-thrombin IgG with AuNPs followed by silver enhancement a greater passive signal enhancement in comparison to the membrane blockage has been observed. The contribution of both electrostatic/steric effects in this blockage due to the subsequent formation of the aptamer-thrombin complex and the final sandwich assay is investigated. The efficiency of the system is also monitored by microscopic techniques. The resulted biosensing system allows detecting thrombin spiked in whole blood at very low levels (LOD 1.8 ng mL(-1)) which are within the range of clinical interest for the diagnostic of coagulation abnormalities as well as pulmonary metastasis.

Design, preparation, and evaluation of a fixed-orientation antibody/gold-nanoparticle conjugate as an immunosensing label

Herein, we describe the development of a new, highly efficient label for immunosensing comprising an antibody/PEGylated gold-nanoparticle (AuNP) conjugate in which the antibody molecules are bound to the AuNP surface in a fixed orientation. Our method exploits the high density of positive charges on the major plane of antibodies that exists when the pH of the solution is lower than the isoelectric point of the antibody; the antibody molecules interact with the negatively charged AuNP surface through their major plane, enabling the antigen binding sites to move freely and therefore to reach maximum accessibility. This directed ionic interaction is reinforced by the formation of a peptide bond between the amino group of the Lys residues in the antibodies and the carboxylic groups of the PEGylated-AuNP surface via EDC chemistry. Electrochemical analyses revealed that the fixed-orientation conjugate offers a limit of detection that is 1 order of magnitude lower than that of a randomly oriented label. The performance of the new conjugate as an immunosensing label was assessed for the quantitative detection of IgG in human serum.

Detection of Circulating Cancer Cells Using Electrocatalytic Gold Nanoparticles

A rapid cancer cell detection and quantification assay, based on the electrocatalytic properties of gold nanoparticles towards the hydrogen evolution reaction, is described. The selective labeling of cancer cells is performed in suspension, allowing a fast interaction between the gold nanoparticle labels and the target proteins expressed at the cell membrane. The subsequent electrochemical detection is accomplished with small volumes of sample and user-friendly equipment through a simple electrochemical method that generates a fast electrochemical response used for the quantification of nanoparticle-labeled cancer cells. The system establishes a selective cell-detection assay capable of detecting 4 × 10(3) cancer cells in suspension that can be extended to several other cells detection scenarios.