Nikhil Bhalla

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.

Introduction to biosensors

Biosensors are nowadays ubiquitous in biomedical diagnosis as well as a wide range of other areas such as point-of-care monitoring of treatment and disease progression, environmental monitoring, food control, drug discovery, forensics and biomedical research. A wide range of techniques can be used for the development of biosensors. Their coupling with high-affinity biomolecules allows the sensitive and selective detection of a range of analytes. We give a general introduction to biosensors and biosensing technologies, including a brief historical overview, introducing key developments in the field and illustrating the breadth of biomolecular sensing strategies and the expansion of nanotechnological approaches that are now available.

Protein phosphorylation analysis based on proton release detection:potential tools for drug discovery

Phosphorylation is the most important post-translational modification of proteins in eukaryotic cells and it is catalysed by enzymes called kinases. The balance between protein phosphorylation and dephosphorylation is critical for the regulation of physiological processes and its unbalance is the cause of several diseases. Conventional assays used to analyse the kinase activity are limited as they rely heavily on phospho-specific antibodies and radioactive tags. This makes their use impractical for high throughput drug discovery platforms. We have developed two versatile methods to detect the release of protons (H(+)) associated with the protein phosphorylation catalysed by kinases. The first approach is based on the pH-sensitive response of oxide-semiconductor interfaces and the second method detects the pH changes in phosphorylation reaction using a commercial micro-pH electrode. The proposed methods successfully detected phosphorylation of myelin basic protein by PKC-α kinase. These techniques can be readily adopted for multiplexed arrays and high throughput analysis of kinase activity, which will represent an important innovation in biomedical research and drug discovery.

Optimisation and Characterisation of Anti-Fouling Ternary SAM Layers for Impedance-Based Aptasensors

An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples.

Protein phosphorylation detection using dual-mode field-effect devices and nanoplasmonic sensors

Phosphorylation by kinases is an important post-translational modification of proteins. It is a critical control for the regulation of vital cellular activities, and its dysregulation is implicated in several diseases. A common drug discovery approach involves, therefore, time-consuming screenings of large libraries of candidate compounds to identify novel inhibitors of protein kinases. In this work, we propose a novel method that combines localized surface plasmon resonance (LSPR) and electrolyte insulator semiconductor (EIS)-based proton detection for the rapid identification of novel protein kinase inhibitors. In particular, the selective detection of thiophosphorylated proteins by LSPR is achieved by changing their resonance properties via a pre-binding with gold nanoparticles. In parallel, the EIS field-effect structure allows the real-time electrochemical monitoring of the protein phosphorylation by detecting the release of protons associated with the kinases activity. This innovative combination of both field-effect and nanoplasmonic sensing makes the detection of protein phosphorylation more reliable and effective. As a result, the screening of protein kinase inhibitors becomes more rapid, sensitive, robust and cost-effective.

Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay

This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems) was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead-protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.

Aptamer-based Field-Effect Biosensor for Tenofovir Detection

During medical treatment it is critical to maintain the circulatory concentration of drugs within their therapeutic range. A novel biosensor is presented in this work to address the lack of a reliable point-of-care drug monitoring system in the market. The biosensor incorporates high selectivity and sensitivity by integrating aptamers as the recognition element and field-effect transistors as the signal transducer. The drug tenofovir was used as a model small molecule. The biointerface of the sensor is a binary self-assembled monolayer of specific thiolated aptamer and 6-mercapto-1-hexanol (MCH), whose ratio was optimized by electrochemical impedance spectroscopy measurements to enhance the sensitivity towards the specific target. Surface plasmon resonance, performed under different buffer conditions, shows optimum specific and little non-specific binding in phosphate buffered saline. The dose-response behavior of the field-effect biosensor presents a linear range between 1 nM and 100 nM of tenofovir and a limit of detection of 1.2 nM. Two non-specific drugs and one non-specific aptamer, tested as stringent control candidates, caused negligible responses. The applications were successfully extended to the detection of the drug in human serum. As demonstrated by impedance measurements, the aptamer-based sensors can be used for real-time drug monitoring.

Inexpensive and fast pathogenic bacteria screening using field-effect transistors

While pathogenic bacteria contribute to a large number of globally important diseases and infections, current clinical diagnosis is based on processes that often involve culturing which can be time-consuming. Therefore, innovative, simple, rapid and low-cost solutions to effectively reduce the burden of bacterial infections are urgently needed. Here we demonstrate a label-free sensor for fast bacterial detection based on metal-oxide-semiconductor field-effect transistors (MOSFETs). The electric charge of bacteria binding to the glycosylated gates of a MOSFET enables quantification in a straightforward manner. We show that the limit of quantitation is 1.9×10(5) CFU/mL with this simple device, which is more than 10,000-times lower than is achieved with electrochemical impedance spectroscopy (EIS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF) on the same modified surfaces. Moreover, the measurements are extremely fast and the sensor can be mass produced at trivial cost as a tool for initial screening of pathogens.

Large‐Scale Nanophotonic Structures for Long‐Term Monitoring of Cell Proliferation

Innovative sensing materials have enabled the discovery of cell biology principles at the nanoscale. In order to evaluate cell behavior and responses, it is necessary to accurately monitor cell proliferation. However, it remains challenging to develop nanomaterials possessing pertinent properties for sensing, while ensuring long‐term cell survival and unaltered cellular responses. This work develops highly sensitive, large‐scale, and biocompatible nanoplasmonic biosensors for long‐term monitoring of cell proliferation, reported for the first time. The nanoplasmonic sensor consists of mushroom‐like structures, with stems of silicon dioxide and caps of gold covering a total surface area of 18.75 cm2. The localized surface plasmons on the nanomushroom caps are exploited to monitor proliferating fibroblast cells. Changes in nanoplasmonic resonances of the nanomushrooms are directly proportional to the number of cells that bind to them. Fibroblast proliferation is successfully monitored for 7 d, demonstrating remarkable biocompatibility of the nanomushroom substrates. These nanomushroom substrates preserve cell viability and serve as a label‐free platform for long‐term monitoring of cell proliferation. These results also open new opportunities in developing standard cell assays without chemical labels to detect cellular responses at the nanoscale.

Plasma-Assisted Large-Scale Nanoassembly of Metal–Insulator Bioplasmonic Mushrooms

Large-scale plasmonic substrates consisting of metal-insulator nanostructures coated with a biorecognition layer can be exploited for enhanced label-free sensing by utilizing the principle of localized surface plasmon resonance (LSPR). Most often, the uniformity and thickness of the biorecognition layer determine the sensitivity of plasmonic resonances as the inherent LSPR sensitivity of nanomaterials is limited to 10-20 nm from the surface. However, because of time-consuming nanofabrication processes, there is limited work on both the development of large-scale plasmonic materials and the subsequent surface functionalizing with biorecognition layers. In this work, by exploiting properties of reactive ions in an SF6 plasma environment, we are able to develop a nanoplasmonic substrate containing ∼106/cm2 mushroom-like structures on a large-sized silicon dioxide substrate (i.e., 2.5 cm by 7.5 cm). We further investigate the underlying mechanism of the nanoassembly of gold on glass inside the plasma environment, which can be expanded to a variety of metal-insulator systems. By incorporating a novel microcontact printing technique, we deposit a highly uniform biorecognition layer of proteins on the nanoplasmonic substrate. The bioplasmonic assays performed on these substrates achieve a limit of detection of 10-17 g/mL (∼66 zM) for biomolecules such as antibodies (∼150 kDa). Our simple nanofabrication procedure opens new opportunities in fabricating versatile bioplasmonic materials for a wide range of biomedical and sensing applications.