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

Electrochemical detection of glycan and protein epitopes of glycoproteins in serum

Aberrant protein glycosylation is associated with a range of pathological conditions including cancer and possesses diagnostic importance. Translation of glycoprotein biomarkers will be facilitated by the development of a rapid and sensitive analytical platform that simultaneously interrogates both the glycan and protein epitopes of glycoproteins in body fluids such as serum or saliva. To this end, we developed an electrochemical biosensor based on the immobilization of a lectin on the gold electrode surface to recognize/capture a target glycan epitope conjugated to glycoproteins, followed by detection of the protein epitope using a target protein-specific antibody. Electrochemical signals are generated by label-free voltammetric or impedimetric interrogation of a ferro/ferricyanide redox couple (e.g. [Fe(CN)(6)](3-/4-)) on the sensing surface, where the change in voltammetric current or interfacial electron transfer resistance was measured. The detection system was demonstrated using the model glycoprotein chicken ovalbumin with Sambucus nigra agglutinin type I (SNA lectin), and exhibits femtomolar sensitivity in the background of diluted human serum. The results obtained in this proof-of-concept study demonstrate the possibility of using electrochemical detection for developing cheap point-of-care diagnostics with high specificity and sensitivity for blood glycoprotein biomarkers

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

Simple, sensitive and inexpensive regional DNA methylation detection methodologies are imperative for routine patient diagnostics. Herein, we describe eMethylsorb, an electrochemical assay for quantitative detection of regional DNA methylation on a single-use and cost-effective screen-printed gold electrode (SPE-Au) platform. The eMethylsorb approach is based on the inherent differential adsorption affinity of DNA bases to gold (i.e. adenine > cytosine ≥ guanine > thymine). Through bisulfite modification and asymmetric PCR of DNA, methylated and unmethylated DNA in the sample becomes guanine-enriched and adenine-enriched respectively. Under optimized conditions, adenine-enriched unmethylated DNA (higher affinity to gold) adsorbs more onto the SPE-Au surface than methylated DNA. Higher DNA adsorption causes stronger coulombic repulsion and hinders reduction of ferricyanide [Fe(CN)]ions on the SPE-Au surface to give a lower electrochemical response. Hence, the response level is directly proportional to the methylation level in the sample. The applicability of this methodology was tested by detecting the regional methylation status in a cluster of eight CpG sites within the engrailed (EN1) gene promoter of the MCF7 breast cancer cell line. A 10% methylation level sensitivity with good reproducibility (RSD = 5.8%, n = 3) was achieved rapidly in 10 min. Furthermore, eMethylsorb also has advantages over current methylation assays such as being inexpensive, rapid and does not require any electrode surface modification. We thus believe that the eMethylsorb assay could potentially be a rapid and accurate diagnostic assay for point-of-care DNA methylation analysis

Detection of regional DNA methylation using DNA-graphene affinity interactions

We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl(-1) of DNA with no sequencing requirement

Application of ionic liquids in electrochemical sensing systems

Since 1992, when the room temperature ionic liquids (ILs) based on the 1-alkyl-3-methylimidazolium cation were reported to provide an attractive combination of an electrochemical solvent and electrolyte, ILs have been widely used in electrodeposition, electrosynthesis, electrocatalysis, electrochemical capacitor, and lithium batteries. However, it has only been in the last few years that electrochemical biosensors based on carbon ionic liquid electrodes (CILEs) and IL-modified macrodisk electrodes have been reported. However, there are still a lot of challenges in achieving IL-based sensitive, selective, and reproducible biosensors for high speed analysis of biological and environmental compounds of interest. This review discusses the principles of operation of electrochemical biosensors based on CILEs and IL/composite-modified macrodisk electrodes. Subsequently, recent developments and major strategies for enhancing sensing performance are discussed. Key challenges and opportunities of IL-based biosensors to further development and use are considered. Emphasis is given to direct electron-transfer reaction and electrocatalysis of hemeproteins and enzyme-modified composite electrodes. © 2010 Elsevier B.V. All rights reserve

Trace analysis of DNA: Preconcentration, separation, and electrochemical detection in microchip electrophoresis using Au nanoparticles

We have developed a simple and sensitive on-chip preconcentration, separation, and electrochemical detection (ED) method for trace analysis of DNA. The microchip comprised of three parallel channels: the first two are for the field-amplified sample stacking and subsequent field-amplified sampled injection steps, while the third one is for the microchip gel electrophoresis (MGE) with ED (MGE-ED). To improve preconcentration and separation performances of the method, the stacking and separation buffers containing the hydroxypropyl cellulose (HPC) matrix were modified with gold nanoparticles (AuNPs). The formation of AuNPs and HPC/AuNP-modified buffers were characterized by UV−visible spectroscopy and TEM experiments. The conducting polymer-modified electrode was also modified with AuNPs to enhance detection performances of the electrode. The conducting polymer/AuNP layers act as electrocatalysts for the direct detection of DNA based on their oxidation in a solution phase. The total sensitivity was improved by 25000-fold when compared with a conventional MGE-ED analysis. The calibration plots were linear (r2 = 0.9993) within the range of 0.003−1.0 pg/μL for a 20-bp DNA sample. The sensitivity was 0.20 nA/(fg/μL), with a detection limit of 5.7 amol in a 50-μL sample, based on S/N = 3. The applicability of the method for the analysis of 13 fragments present in a 100-bp DNA ladder was successfully demonstrated

Microchip and capillary electrophoresis using nanoparticles

Explores the latest applications arising from the intersection of nanotechnology and microfluidics In the past two decades, microfluidics research has seen phenomenal growth, with many new and emerging applications in fields ranging from chemistry, physics, and biology to engineering. With the emergence of nanotechnology, microfluidics is currently undergoing dramatic changes, embracing the rising field of nanofluidics. This volume reviews the latest devices and applications stemming from the merging of nanotechnology with microfludics in such areas as drug discovery, bio-sensing, catalysis, electrophoresis, enzymatic reactions, and nanomaterial synthesis. Each of the ten chapters is written by a leading pioneer at the intersection of nanotechnology and microfluidics. Readers not only learn about new applications, but also discover which futuristic devices and applications are likely to be developed. Topics explored in this volume include: * New lab-on-a-chip systems for drug delivery * Integration of microfluidics with nanoneuroscience to study the nervous system at the single-cell level * Recent applications of nanoparticles within microfluidic channels for electrochemical and optical affinity biosensing * Novel microfluidic approaches for the synthesis of nanomaterials * Next-generation alternative energy portable power devices References in each chapter guide readers to the primary literature for further investigation of individual topics. Overall, scientists, researchers, engineers, and students will not only gain a new perspective on what has been done, but also the nanotechnology tools they need to develop the next generation of microfluidic devices and applications. Microfluidic Devices for Nanotechnology is a two-volume publication, the first ever to explore the synergies between microfluidics and nanotechnology. The first volume covers fundamental concepts; this second volume examines applications

"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

Detecting DNA methylation for cancer diagnostics and prognostics

DNA methylation is an epigenetic process that has roles in many normal cellular processes and dysregulation of which can result in diseases such as cancer. The most well studied form of mammalian DNA methylation is the addition of a methyl group to the number 5 carbon of cytosine in CpG dinucleotides. Gene regulatory elements, such as gene promoters and enhancers associated with dense CpGs, are sensitive to DNA methylation silencing. Multiple studies have shown that promoter methylation profiling of cancer genes may be useful as biomarkers of cancer. DNA methylation information has the potential to provide information on a patient's cancer subtype, treatment response and prognosis. Current DNA methylation detection techniques can be broadly grouped into sodium bisulfite, restriction enzyme and methylated DNA enrichment based techniques. However, techniques for detection DNA methylation have largely been tailored to research purposes and may not be well suited for routine clinical use. In this chapter, some of these common DNA methylation detection methods are reviewed for their suitability in diagnostics. Also discussed are ways how some of these techniques may be or have been adapted for clinical and point-of-care applications. Emerging techniques that have evolved from classical research methods are also introduced