Pedro Estrela

Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy

The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1-9) x10(12)/cm2. The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 x 10(12)/cm2, electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.

Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes

DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.

Carbon Nanostructure-Based Field-Effect Transistors for Label-Free Chemical/Biological Sensors

Over the past decade, electrical detection of chemical and biological species using novel nanostructure-based devices has attracted significant attention for chemical, genomics, biomedical diagnostics, and drug discovery applications. The use of nanostructured devices in chemical/biological sensors in place of conventional sensing technologies has advantages of high sensitivity, low decreased energy consumption and potentially highly miniaturized integration. Owing to their particular structure, excellent electrical properties and high chemical stability, carbon nanotube and graphene based electrical devices have been widely developed for high performance label-free chemical/biological sensors. Here, we review the latest developments of carbon nanostructure-based transistor sensors in ultrasensitive detection of chemical/biological entities, such as poisonous gases, nucleic acids, proteins and cells.

Point-of-Care Diagnostics in Low Resource Settings: Present Status and Future Role of Microfluidics

The inability to diagnose numerous diseases rapidly is a significant cause of the disparity of deaths resulting from both communicable and non-communicable diseases in the developing world in comparison to the developed world. Existing diagnostic instrumentation usually requires sophisticated infrastructure, stable electrical power, expensive reagents, long assay times, and highly trained personnel which is not often available in limited resource settings. This review will critically survey and analyse the current lateral flow-based point-of-care (POC) technologies, which have made a major impact on diagnostic testing in developing countries over the last 50 years. The future of POC technologies including the applications of microfluidics, which allows miniaturisation and integration of complex functions that facilitate their usage in limited resource settings, is discussed The advantages offered by such systems, including low cost, ruggedness and the capacity to generate accurate and reliable results rapidly, are well suited to the clinical and social settings of the developing world.

Aptamer-MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen

This study reports the design and evaluation of a new synthetic receptor sensor based on the amalgamation of biomolecular recognition elements and molecular imprinting to overcome some of the challenges faced by conventional protein imprinting. A thiolated DNA aptamer with established affinity for prostate specific antigen (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, its binding conformation, and to localise the PSA binding sites at the sensor surface. Following removal of PSA, 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 recognition properties superior to that of aptamer alone. Electrochemical impedance spectroscopy (EIS) was used to evaluate subsequent rebinding of PSA to the apta-MIP surface. The apta-MIP sensor showed high sensitivity with a linear response from 100pg/ml to 100ng/ml of PSA and a limit of detection of 1pg/ml, which was three-fold higher than aptamer alone sensor for PSA. Furthermore, the sensor demonstrated low cross-reactivity with a homologous protein (human Kallikrein 2) and low response to human serum albumin (HSA), suggesting possible resilience to the non-specific binding of serum proteins.

Label-free sub-picomolar protein detection with field-effect transistors

Proteins mediate the bulk of biological activity and are powerfully assayed in the diagnosis of diseases. Protein detection relies largely on antibodies, which have significant technical limitations especially when immobilized on two-dimensional surfaces. Here, we report the integration of peptide aptamers with extended gate metal-oxide-semiconductor field-effect transistors (MOSFETs) to achieve label-free sub-picomolar target protein detection. Specifically, peptide aptamers that recognize highly related protein partners of the cyclin-dependent kinase (CDK) family are immobilized on the transistor gate to enable human CDK2 to be detected at 100 fM or 5 pg/mL, well within the clinically relevant range. The target specificity, ease of fabrication, and scalability of these FET arrays further demonstrate the potential application of the multiplexable field effect format to protein sensing.

Localized Surface Plasmon Resonance as a Biosensing Platform for Developing Countries

The discovery of the phenomena known as localized surface plasmon resonance (LSPR) has provided the basis for many research areas, ranging from materials science to biosensing. LSPR has since been viewed as a transduction platform that could yield affordable, portable devices for a multitude of applications. This review aims to outline the potential applications within developing countries and the challenges that are likely to be faced before the technology can be effectively employed.

Chemical and biological sensors using polycrystalline silicon TFTs

Over the past three decades effort has been devoted to exploit the field-effect mechanism in chemical and biological sensors, due to the potential of these devices to provide large arrays of sensors that are label-free, low-cost, disposable and can be easily integrated in portable instrumentation. Most of this work concerned the development of ion-sensitive field-effect transistors. More recently, field-effect devices have been investigated for the detection of DNA hybridization and protein interactions. Of particular interest is the use of polycrystalline silicon thin film transistors. This technology is inherently low cost and yet capable of providing complex single-use microarrays.

Polycrystalline silicon ion sensitive field effect transistors

We report the operation of polycrystalline silicon ion sensitive field effect transistors. These devices can be fabricated on inexpensive disposable substrates such as glass or plastics and are, therefore, promising candidates for low cost single-use intelligent multisensors. In this work we have developed an extended gate structure with a Si3N4 sensing layer. Nearly ideal pH sensitivity (54mV∕pH) and stable operation have been achieved. Temperature effects have been characterized. A penicillin sensor has been fabricated by functionalizing the sensing area with penicillinase. The sensitivity to penicillin G is about 10mV∕mM, in solutions with concentration lower than the saturation value, which is about 7 mM.

Electrochemical biosensors and nanobiosensors.

Electrochemical techniques have great promise for low-cost miniaturised easy-to-use portable devices for a wide range of applications–in particular, medical diagnosis and environmental monitoring. Different techniques can be used for biosensing, with amperometric devices taking the central role due to their widespread application in glucose monitoring. In fact, glucose biosensing takes an approximately 70% share of the biosensor market due to the need for diabetic patients to monitor their sugar levels several times a day, making it an appealing commercial market. In this review, we present the basic principles of electrochemical biosensor devices. A description of the different generations of glucose sensors is used to describe in some detail the operation of amperometric sensors and how the introduction of mediators can enhance the performance of the sensors. Electrochemical impedance spectroscopy is a technique being increasingly used in devices due to its ability to detect variations in resistance and capacitance upon binding events. Novel advances in electrochemical sensors, due to the use of nanomaterials such as carbon nanotubes and graphene, are presented as well as future directions that the field is taking.