Continuous monitoring of molecular biomarkers in microfluidic devices.

The ability to monitor molecular targets is crucial in fields ranging from healthcare to industrial processing to environmental protection. Devices employing biomolecules to achieve this goal are called biosensors. Over the last half century researchers have developed dozens of different biosensor approaches. In this chapter we analyze recent advances in the biosensing field aiming at adapting these to the problem of continuous molecular monitoring in complex sample streams, and how the merging of these sensors with lab-on-a-chip technologies would be beneficial to both. To do so we discuss (1) the components that comprise a biosensor, (2) the challenges associated with continuous molecular monitoring in complex sample streams, (3) how different sensing strategies deal with (or fail to deal with) these challenges, and (4) the implementation of these technologies into lab-on-a-chip architectures.

Rapid on-chip apoptosis assay on human carcinoma cells based on annexin-V/quantum dot probes.

Despite all the efforts made over years to study the cancer expression and the metastasis event, there is not a clear understanding of its origins and effective treatment. Therefore, more specialized and rapid techniques are required for studying cell behaviour under different drug-based treatments. Here we present a quantum dot signalling-based cell assay carried out in a segmental microfluidic device that allows studying the effect of anti-cancer drugs in cultured cell lines by monitoring phosphatidylserine translocation that occurs in early apoptosis. The developed platform combines the automatic generation of a drug gradient concentration, allowing exposure of cancer cells to different doses, and the immunolabeling of the apoptotic cells using quantum dot reporters. Thereby a complete cell-based assay for efficient drug screening is performed showing a clear correlation between drug dose and amount of cells undergoing apoptosis.

Water Activated Graphene Oxide Transfer Using Wax Printed Membranes for Fast Patterning of a Touch Sensitive Device.

We demonstrate a graphene oxide printing technology using wax printed membranes for the fast patterning and water activation transfer using pressure based mechanisms. The wax printed membranes have 50 µm resolution, longtime stability and infinite shaping capability. The use of these membranes complemented with the vacuum filtration of graphene oxide provides the control over the thickness. Our demonstration provides a solvent free methodology for printing graphene oxide devices in all shapes and all substrates using the roll-to-roll automatized mechanism present in the wax printing machine. Graphene oxide was transferred over a wide variety of substrates as textile or PET in between others. Finally, we developed a touch switch sensing device integrated in a LED electronic circuit.

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry.

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.

The use of quantum dots for immunochemistry applications.

Immunocytochemistry and histochemistry are two most valuable immunochemistry techniques routinely used in biological laboratories. These techniques rely on the use of antibodies to label epitopes of interest in cells. At present, there is a wide range of commercially available organic dyes for labeling antibodies. However, limited extinction coefficients of organic dyes often make it difficult to achieve the optimal exposure and therefore fluorescence detection limit. Quantum dots (QDs) are fluorescent semiconductor nanocrystals which are advantageous over the organic fluorescent dyes in many aspects, principally their long-term luminescence stability, high brightness, and multicolor detection. Here, we describe the use of QDs for immunocytochemistry applications. We used three different antibodies-anti-ß-tubulin monoclonal antibody for visualizing the microtubule network, the GM130 antibody for staining the Golgi complex, and the EEA1 antibody for detecting the endosomal system. We use the anti-mouse IgG antibody directly conjugated to QD655 quantum dots or anti-mouse IgG conjugated to biotin for tertiary detection with streptavidin-conjugated QD655.

Signal enhancement in antibody microarrays using quantum dots nanocrystals: application to potential Alzheimer's disease biomarker screening.

The performance of cadmium-selenide/zinc-sulfide (CdSe@ZnS) quantum dots (QDs) and the fluorescent dye Alexa 647 as reporter in an assay designed to detect apolipoprotein E (ApoE) has been compared. The assay is a sandwich immunocomplex microarray that functions via excitation by visible light. ApoE was chosen for its potential as a biomarker for Alzheimer's disease. The two versions of the microarray (QD or Alexa 647) were assessed under the same experimental conditions and then compared to a conventional enzyme-linked immunosorbent assay (ELISA) targeting ApoE. The QDs proved to be highly effective reporters in the microarrays, although their performance strongly varied in function of the excitation wavelength. At 633 nm, the QD microarray gave a limit of detection (LOD) of ~247 pg mL(-1); however, at an excitation wavelength of 532 nm, it provided a LOD of ~62 pg mL(-1), five times more sensitive than that of the Alexa microarray (~307 pg mL(-1)) and seven times more than that of the ELISA (~470 pg mL(-1)). Finally, serial dilutions from a human serum sample were assayed with high sensitivity and acceptable precision and accuracy.

QDs versus Alexa: reality of promising tools for immunocytochemistry.

BACKGROUND: The unique photonic properties of the recently developed fluorescent semiconductor nanocrystals (QDs) have made them a potential tool in biological research. However, QDs are not yet a part of routine laboratory techniques. Double and triple immunocytochemistries were performed in HeLa cell cultures with commercial CdSe QDs conjugated to antibodies. The optical characteristics, due to which QDs can be used as immunolabels, were evaluated in terms of emission spectra, photostability and specificity. RESULTS: QDs were used as secondary and tertiary antibodies to detect beta-tubulin (microtubule network), GM130 (Golgi complex) and EEA1 (endosomal system). The data obtained were compared to homologous Alexa Fluor 594 organic dyes. It was found that QDs are excellent fluorochromes with higher intensity, narrower bandwidth values and higher photostability than Alexa dyes in an immunocytochemical process. In terms of specificity, QDs showed high specificity against GM130 and EEA1 primary antibodies, but poor specificity against beta-tubulin. Alexa dyes showed good specificity for all the targets tested. CONCLUSION: This study demonstrates the great potential of QDs, as they are shown to have superior properties to Alexa dyes. Although their specificity still needs to be improved in some cases, QDs conjugated to antibodies can be used instead of organic molecules in routine immunocytochemistry.