Quantum dot encoded magnetic beads for multiplexed fluorescence biosensing

In recent years, the use of encoded beads has received considerable attention due to their potential for measuring multiple analytes in solution.(1-4) This can be achieved without the need for knowledge of their spatial position, as in the case of microarray technology. Encoded bead technology also relies on the solution kinetics rather than diffusion to a fixed surface as in the case of microarray technology, offering the possibility of developing rapid high throughput screening methods. This thesis describes the production, characterisation and application of quantum dot encoded beads prepared using layer-by-layer assembly of different colour quantum dots around a magnetic bead. To achieve this, two different strategies were used to make “coloured” barcodes. The first strategy used thiol chemistry to immobilise quantum dots in a layer-by-layer assembly onto magnetic beads whereas the second strategy uses the interaction between quantum dot-biotin and quantum dot-streptavidin conjugates to create constructs on the magnetic bead surface. The development of both of these immobilisation strategies was characterisation using X-ray photoelectron spectroscopy and fluorescence spectroscopy of immobilised quantum dot structures onto a plain glass substrate. After the preparation of encoded beads, they were characterised using single bead fluorescence spectroscopy. It was found that attempts to prepare barcodes by layer-by-layer assembly of CdSe/ZnS quantum dots using thiol chemistry onto magnetic beads did not comply with the necessary barcode characteristics i.e., different colour coded beads could not be distinguished from each other. However, the encoded beads prepared using layer-by-layer assembly of quantum dot-biotin and quantum dot-streptavidin conjugates onto streptavidin coated magnetic beads gave distinct multicolour coded bead spectra. These barcodes were characterised in terms of different spectral responses, stability at raised temperatures, stability in biotin solutions, and long-term stability after storage. Encoded beads prepared using layer-by-layer assembly of quantum dot-biotin and quantum dot-streptavidin conjugates onto streptavidin coated magnetic beads were then used to develop multiplexed immunoassays. Four different barcodes were prepared and used to perform model multiplexed immunoassays. The barcodes were identified upon the basis of different spectral response measured using single bead fluorescence spectroscopy. Finally, a quantitative immunoassay for human IgG was performed using these barcodes, which showed that different concentrations of human IgG can be determined in solution

Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing

Exosomes show promise as non-invasive biomarkers for cancers, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for highly specific capture and detection of multiple exosome targets using a tuneable alternating current electrohydrodynamic (ac-EHD) methodology - referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow which enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and exhibits a 5-fold detection enhancement compared to hydrodynamic flow based assays. The device was also sensitive enough to detect approximately 2750 exosomes/µL (n = 3) and also capable of specifically isolating exosomes from breast cancer patient samples. We believe this approach can potentially find its relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications

Molecular inversion probe-based SPR biosensing for specific, label-free and real-time detection of regional DNA methylation

DNA methylation has the potential to be a clinically important biomarker in cancer. This communication reports a real-time and label-free biosensing strategy for DNA methylation detection in the cancer cell line. This has been achieved by using surface plasmon resonance biosensing combined with the highly specific molecular inversion probe based amplification method, which requires only 50 ng of bisulfite treated genomic DNA

"Drill and fill" lithography for controlled fabrication of 3D platinum electrodes

Reproducible fabrication of three-dimensional (3D) metal electrodes is essential for accurate measurements of analyte in the fields of electroanalytical chemistry and biosensor development. Unfortunately, fabrication of these types of electrodes is complicated by the inherent complexity of current nano-fabrication methodologies. Herein, we report a simple and rapid fabrication method referred to as "drill and fill" lithography that provides control structuring and excellent reproducibility in the fabrication of 3D Pt electrodes with different sizes and shapes. The ability to combine "drill" and "fill" in a single technique using focused ion beam (FIB) lithography enabled precise control of the size and shape of the electrode depending on the amount of deposited metallic Pt. Electrodes were characterized using scanning electron microscopy, cyclic voltammetry, and Faradaic electrochemical impedance spectroscopy. The key innovation of this methodology lies in its simplicity and ability to tune the operating parameters to obtain electrodes of designated size and shapes. We envisage that these electrodes could be ideal for 'on-chip' diagnostic platforms

Electrohydrodynamic removal of non-specific colloidal adsorption at electrode interfaces

This communication reports the use of an electrohydrodynamic surface shear force to selectively manipulate colloid-surface interactions. We demonstrate the selection of strongly (specifically) bound biomolecule-functionalized colloidal beads over more weakly (non-specifically) bound beads using a tuneable alternating current electrohydrodynamic (ac-EHD) force, which drives lateral fluid motion within a few nanometers of an electrode surface. By externally "tuning" the strength of the ac-EHD force, we demonstrate a significant enhancement of capture efficiency for specifically bound colloids, along with a removal of the adsorption of non-specific colloidal beads-a process which may be observed in real-time

"Drill and fill" lithography: fabrication of platinum electrodes and their use in label-free immunosensing

We report a new fabrication method referred to as 'drill and fill' lithography that provides control structuring and excellent reproducibility in the production of patterned platinum electrodes in a chip format, and demonstrate their sensing abilities for the label-free immunodetection of the HER-2 (human epidermal growth factor receptor-2) antigen

Highly selective metal–organic framework textile humidity sensor

The increase in demand and popularity of smart textiles brings new and innovative ideas to develop a diverse range of textile-based devices for our daily life applications. Smart textile-based sensors (TEX sensors) become attractive due to the potential to replace current solid-state sensor devices with flexible and wearable devices. We have developed a smart textile sensor for humidity detection using a metal–organic framework (MOF) as an active thin-film layer. We show for the first time the use of the Langmuir–Blodgett (LB) technique for the deposition of a MIL-96(Al) MOF thin film directly onto the fabrics containing interdigitated textile electrodes for the fabrication of a highly selective humidity sensor. The humidity sensors were made from two different types of textiles, namely, linen and cotton, with the linen-based sensor giving the best response due to better coverage of MOF. The TEX sensor showed a reproducible response after multiple cycles of measurements. After 3 weeks of storage, the sensor showed a moderate decrease in response. Moreover, TEX sensors showed a high level of selectivity for the detection of water vapors in the presence of several volatile organic compounds (VOCs). Interestingly, the selectivity is superior to some of the previously reported MOF-coated solid-state interdigitated electrode devices and textile sensors. The method herein described is generic and can be extended to other textiles and coating materials for the detection of toxic gases and vapors.M.A.A. acknowledges the support of Ministerio de Educación from the Spanish Government under a FPU grant (Formación de Profesorado Universitario, FPU14/05367) and a short-term mobility FPU grant (EST18/00291) and of King Abdullah University of Science and Technology and Advanced Membranes and Porous Materials Center under the Visiting Student Program.Peer reviewe

Tuneable surface shear forces to physically displace nonspecific molecules in protein biomarker detection

We report a simple method to remove nonspecifically adsorbed species from sensor surface and also improve the detection sensitivity of the sensor using tuneable alternating current (ac) electrohydrodynamics (ac-EHD) forces. These forces generated within few nanometers of an electrode surface (i.e., double layer) engender fluid flow within a serpentine channel containing a long array of the asymmetric electrode pairs, and can easily be tuned externally by changing the frequency and amplitude of the ac-EHD field. Under the optimized experimental conditions, we achieved a 3.5-fold reduction in nonspecific adsorption of non-target proteins with a 1000-fold enhancement in detection sensitivity of the device for the analysis of human epidermal growth factor receptor 2 (HER2) protein spiked in serum. This approach can be applicable in diverse fields including biosensors, cellular and molecular separation systems and biomedical applications to remove/reduce nonspecific adsorption of molecular and cellular species