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RESEARCH HIGHLIGHTS

March 12, 2017

DNA Biosensor Based on PCB Technology

Since the introduction of the Lab-on-Chip concept in the 90's, the global excitement for this revolutionary technology, promising to shrink complete biomedical laboratories in a few centimeter chips, has been increasing exponentially. One of the main driving applications for this technology is its exploitation for transferring medical diagnostic tests from centralized clinical laboratories at the Point-of-Care, owing to its tantalizing advantages over conventional laboratory tests: rapid response time, miniaturized sample volumes, automation, portability. The extensive research efforts from the increasingly growing global Lab-on-a-Chip (LoC) community over these years have proven these advantages and the feasibility of having such devices in real-life use (eg home blood glucose tests, home pregnancy tests). However, manufacturability issues have not been successfully addressed until now for many research prototypes. In our case, we are trying to fundamentally address these commercialization bottlenecks, exploiting our biosensor technology under development. Printed Circuit Board (PCB) fabrication allows the low-cost mass fabrication of the platforms, while molecular identification can be applied for various clinically relevant biomarkers, ranging from bacterial infections to cancer diagnosis. Dr Despina Moschou joined the University of Bath as a 50th anniversary Prize Fellow in Bioelectronics in 2016. Her role furthers expands on research in Lab-on-Chip devices for biomedical applications based on the PCB technology. Together with Despina at the University of Bath, PCB technology coupled with microfluidics is currently being investigated to develop novel sensitive biosensors for DNA/RNA detection. 

November 07, 2016

Electro-engineered polymeric films (fast, easy and robust)-AMACR aptasensor

 

Prostate cancer (PCa) is a leading cause of cancer-related mortality among men worldwide. Whilst prostate specific antigen (PSA) remains the benchmark for prostate cancer diagnosis, its use is considered unreliable due to lack of specificity. One of the potential biomarkers that has been gaining significant attention is α-methylacyl-CoA racemase (AMACR; P504S). AMACR has been shown to have higher sensitivity and specificity than PSA for PCa identification in humoral immune assays, especially those with intermediate levels of PSA.

 

The research group from National Taiwan University developed for the first time an aptamer specific to AMACR. Joining together with National Taiwan University, and the Department of Pharmacy & Pharmacology from the University of Bath, we developed an electrochemical AMACR aptasensor based on electro-patterned polyethylene glycol (PEG) on polypyrrole (PPy) film. PEG as a polymer has been widely used to develop biosensors, due to its anti-fouling properties. However, it becomes difficult to employ PEG in an electrochemical platform due to its electrical insulation properties. We propose a one-step, simple strategy to deposit PEG by using an organic conducting polymers such as PPy as a foundation surface. 

May 14, 2015

 

To develop sensitive and selective electrochemical aptasensors, electrode surface chemistry is one of the biggest fields of investigation. Research worldwide is typically focussed on finding the most suitable recognition platform to give a stable organization to the sensor interface leading to optimised binding efficiency and signal outcome. In collaboration with the Slovak Academy of Sciences (Slovakia) and Rhodes University (South Africa), we have characterised three types of apatsensors for prostate cancer biomarker detection. 

 

We have compared different surface chemistries based on planor gold surface. Two types of PSA aptasensor were fabricated by immobilizing (i) a self-assembled monolayer comprising of 6-mercaptohexanol (MCH) and thiolated-DNA aptamer and (ii) 11-mercaptoundecanoic acid for covalent immobilization of amine terminated DNA aptamers and sulfo-betaine terminated thiol as an antifouling agent on a polycrystalline gold surface. 

 

A new molecule called thiol terminated sulfo-betaine has been used for the development of a PSA aptasensor. It contains both positive and negative charges and acts like a zwitterion. It has been previously reported as a potential candidate to reduce non-specific binding and increase the sensitivity of the sensor performance. 

 

Taking a previous system based on an impedimetric aptasensor which used a planar gold surface with co-immobilised DNA aptamer / 6-mercapto-1-hexanol (MCH) probe layer, we show how sensitivity can be significantly improved by the addition of a single fabrication step to attach AuNPs to the planar gold electrode.

August 20, 2015

Aptamer and Molecular Imprinting - A New Milestone

 

Together with Cardiff University, we developed novel hybrid molecularly imprinted DNA aptamers (Aptamer-MIP hybrid). Hybrid imprinting, which integrates a bioreceptor with established affinity for a template molecule within a molecularly imprinted polymer, is a relatively a new approach to generating synthetic receptors for complex biomolecules such as proteins. It allows for greater control of template orientation and polymerisation conditions, while the bioreceptors act synergestically with the polymer conferring increased sensitivity and selectivity.

 

This new synthetic receptor sensor based on the combination of DNA aptamers and molecular imprinting have the potential to overcome some of the challenges faced by both conventional molecular imprinting and DNA aptamers. A thiolated DNA aptamer with established affinity for PSA was complexed with PSA prior to being immobilised on the surface of a gold electrode. Controlled electropolymerisation of dopamine around the complex served to both entrap the complex, holding the aptamer in, or near to, it’s binding conformation, and to localise the PSA binding sites at the sensor surface. The PSA was then removed from the template. It was proposed that the molecularly imprinted polymer (MIP) cavity would act synergistically with the embedded aptamer to form a hybrid receptor (apta–MIP), displaying highly selective aptamer pockets to capture the cancer biomarker.The sensors can be measured with an electrochemical impedance spectroscopy technique or by the use of field-effect transistors – both techniques provided excellent sensitivity and selectivity for a protein cancer biomarker. Critically, both techniques can be easily integrated into a low-cost, small-footprint biosensing platform making them ideal candidates for point-of-need applications. 

December 19, 2015

A new approach to glycoprofiling for enhanced specificity and sensitivity- Optical Aptasensor

 

Classical antibody-based ELISA sandwich immunoassays are widely utilized in medical diagnostic applications. However, issues with antibody cross reactivity and reproducibility limit the accuracy, sensitivity and selectivity of ELISA assays. Replacing the antibodies in a classical ELISA configuration has enabled the development of sophisticated assays which are more robust, reproducible and economical.

Joining together with the teams from INESC MN (Portugal) and Slovak Academy of Sciences (Slovakia) , we developed an aptamer-based ELISA assay study in a microfluidic platform comprising of a sandwich model, where the target protein (Prostate Specific Antigen, PSA, which is a biomarker for Prostate Cancer was used as a case study) is captured by aptamers immobilized covalently in the microfluidic channel. A secondary antibody (Aptamer-Antibody Assay) or a lectin (Aptamer-Lectin Assay) is used to quantify, by chemiluminescence, both the amount of free PSA and its glycosylation levels. Glycoprofiling is of significant importance in diagnosis and prognosis since tumour progression is associated with changes in PSA glycosylation. 

The study addresses how the use of aptamers could be an effective methodology to address current antibody limitations with cross reactivity issues in glycoprofiling. It also shows how aptamers could be used as a potential tool for multi-glycan profiling of biomarkers with high sensitivity and selectivity in a simple microfluidic channel. This approach can be easily extended to a wide range of other biomarkers available for prostate and other cancers. The simultaneous detection of protein cancer biomarkers together with their levels of glycosylation can provide improved diagnosis and prognosis of the disease.

 

Researchers from École Polytechnique Fédérale de Lausanne (EPFL, Switzerland), University of Bath (UK), and University of Applied Sciences Kaiserslautern (Zweibrücken, Germany) came together to develop the first ever-reported electrochemical biosensor based on a memristive effect and DNA aptamers for the detection of prostate cancer biomarker.

 

This novel device is developed to propose a completely new approach in cancer diagnostics. In this study, an affinity-based technique is presented for the detection of the prostate-specific antigen (PSA) using DNA aptamers. The hysteretic properties of memristive silicon nanowires functionalized with these DNA aptamers provide a label-free and ultrasensitive bio detection technique. 

 

DNA aptamers are single-stranded DNA sequences that are developed to bind to its target with high affinity and specificity by undergoing a conformational change. Memory effects are widely appearing in nature, for example, the information gathers stored and retrieved in synaptic connections in the brain, mechanical information stored in the shape of side-chains polymers, and memory effects existing in the molecular organization. EPFL researchers have managed to get around this obstacle by inventing a new detection technique. The trick is to trap the molecules of interest by the blood sample and then detect them in a dry environment, where measurements won’t be disturbed by all the molecules. To do this, the researchers used a Memristor – a new electrical component that can “remember” all the electrical currents that pass through it. The two-terminal Schottky-barrier silicon nanowire devices exhibiting memristive behavior at their electrical response, so-called Memristive Devices when functionalized with aptamers films to obtain Memristive Biosensors for bio-detection purposes. More specifically, the electrical characteristics obtained from the memristive nanowire devices exhibit hysteretic properties and hysteresis loop at zero voltage. However, when biological substances, namely aptamers are present on the devices’ surface the hysteresis appears shifted to different voltage values, and a voltage gap is introduced in the semi-logarithmic current to voltage characteristics after the bio-functionalization process, leading to a label-free bio-detection method of the biological substances. The presence of antigens seems to have a masking contribution to the voltage gap created due to the presence of antibodies on the surface and a construction of the voltage gap occurs with increasing antigen concentration.  

March 15, 2017

Small Molecule (drug) Detection using combination of Aptamers and Field Effect Devices

 

In all medical drug treatments, it is critical to maintain the circulatory concentration of the therapeutic compound in the right range. Together with École Polytechnique Fédérale de Lausanne (EPFL, Switzerland), a  novel biosensor for ultra-small molecules monitoring and more specifically for drug detection (< 900 g mol-1) with high specificity and sensitivity is proposed and characterized in this work. The biosensor achieves both high selectivity and high sensitivity by integrating aptamers as the recognition element and field effect transistor as a signal transducer.

An n-channel metal-oxide semiconductor field effect transistor device (n-MOSFET) with extended gate functionalized with specific DNA aptamer is used in this work to overcome the above-mentioned issues. In order to expose the substrate, the physical gate is extended by connecting it to an external electrode. Then, the substrate is functionalized with specific aptamer to the targeted drug. When the potential applied across the gate and the source (Vgs) is larger than the threshold voltage (Vth) of the device (Vgs >Vth), a conducting channel is formed between the source and the drain. Additionally, if a voltage is applied over the drain and source (Vds>0), then the current (Id) begins to flow through the induced channel under the gate. This condition of the device is known as turn-on state, where the electron-current (Id) enters the drain and exits the source. Any changes across Vgs can modulate the conductivity of the channel and alter the Id. In the case of molecular interactions at the gate of the transistor, such as negatively charged small molecules (for instance drugs) captured by recognition probes (aptamers in this work), the minimum Vgs required to bring the n-MOSFET in the turn-on state is increased (gating effect).

This Aptamer-based Field Effect Transistor (AptaFET), is then proposed for point of care (POC) drug monitoring and successfully tested with potential applications in human plasma and real-time monitoring. Tenofovir was used in this study as a model molecule.

November 08, 2016

Electrochemical MicroRNA Detection

 

MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Together with the University of São Paulo (USP), Brazil, we developed a dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids (PNA) as probes on gold electrode surfaces to capture target miRNAs. PNA was used as it presents many advantages such as neutral charge and higher stability than its biological counterparts. Also, the PNA/miRNA duplex with mismatches is less stable than a DNA/miRNA duplex with the same mismatches. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. 

Such a system was previously developed by the University of Bath using an open circuit potential technique

March 31, 2017

The forgotton Technique (Open Circuit Potential Measurements)

 

At the University of Bath, we developed a sensitive label-free, cost-effective detection system with simultaneous multi-channel measurement of open circuit potential (OCP) variations for the detection of prostate specific antigen (PSA).

The OCP setup was built in-house by Caleb Wong at the University of Bath. The authors believe that the cost of the open circuit potential detection circuit (electronics) for the use in electrochemical detection are cost-effective, and we have implemented this with a flow detection technique. Since the circuit is simple with in-house designed and printable circuits, costs could be kept low. The main components, instrumentation amplifier INA116 (Texas Instruments, US) cost $8.70 each, is used in every channel for the detection of OCP; the analog-to-digital converter (ADC) MAX11040K (Maxim Integrated, USA) costs $13.82 each, is used for every 4 channels. To accurately measure OCP variations, a complete monolithic field-effect transistor (FET)-input ultra-low input bias current instrumentation amplifier is used to form the electronic circuit to measure the variation between a working electrode and a reference electrode. This amplifier electronic system setup provides a differential voltage measurement with high input impedance and low input bias current. Since no current is applied to the electrochemical system, a true and accurate measurement of the variation can be performed. This is the first report on the use of DNA aptamers with an OCP system where we employed a DNA aptamer against PSA. An optimized ratio of anti-PSA DNA aptamer with 6-mercapto-1-hexanol (MCH) was used to fabricate the aptasensor using gold electrodes. The electrodes are hosted in a cell with an automated flow system. The developed system can be further generalized to various other targets using specific probes.

November 18, 2016

Intercalator for Peptide Nucleic Acid (PNA)/DNA Duplex

 

Peptide Nucleic Acid (PNA) are the synthetic analogue of DNA molecules. Unlike a sugar-phosphate backbone of DNA, PNA consists of a peptide backbone, giving it a neutral charge. We report the first intercalator that binds to PNA/DNA duplex. A ferrocenyl intercalator was investigated to develop an electrochemical DNA biosensor employing a peptide nucleic acid (PNA) sequence as a capture probe. After hybridization with single strand DNA sequence, a naphthalene diimide intercalator bearing ferrocene moieties (FND) was introduced to bind with the PNA-DNA duplex and the electrochemical signal of the ferrocene molecules was used to monitor the DNA recognition. Electrochemical impedance spectroscopy was used to characterize the different modification steps. Differential pulse voltammetry was employed to evaluate the electrochemical signal of the FND intercalator related to its interaction with the complementary PNA-DNA hybrid. The ferrocene oxidation peaks were utilized for the target DNA quantification.

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Taiwan
Cardiff
INESC
Germany
Brazil
EPFL
Slovakia
Intercalator
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