Md. Azahar Ali

Microfluidic-integrated biosensors: Prospects for point-of-care diagnostics

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics‐integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro‐ to nano‐liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point‐of‐care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic‐integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.

Highly Sensitive Biofunctionalized Mesoporous Electrospun TiO2 Nanofiber Based Interface for Biosensing

The surface modified and aligned mesoporous anatase titania nanofiber mats (TiO2-NF) have been fabricated by electrospinning for esterified cholesterol detection by electrochemical technique. The electrospinning and porosity of mesoporous TiO2-NF were controlled by use of polyvinylpyrrolidone (PVP) as a sacrificial carrier polymer in the titanium isopropoxide precursor. The mesoporous TiO2-NF of diameters ranging from 30 to 60 nm were obtained by calcination at 470 °C and partially aligned on a rotating drum collector. The functional groups such as -COOH, -CHO etc. were introduced on TiO2-NF surface via oxygen plasma treatment making the surface hydrophilic. Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) were covalently immobilized on the plasma treated surface of NF (cTiO2-NF) via N-ethyl-N0-(3-dimethylaminopropyl carbodiimide) and N-hydroxysuccinimide (EDC-NHS) chemistry. The high mesoporosity (∼61%) of the fibrous film allowed enhanced loading of the enzyme molecules in the TiO2-NF mat. The ChEt-ChOx/cTiO2-NF-based bioelectrode was used to detect esterified cholesterol using electrochemical technique. The high aspect ratio, surface area of aligned TiO2-NF showed excellent voltammetric and catalytic response resulting in improved detection limit (0.49 mM). The results of response studies of this biosensor show excellent sensitivity (181.6 μA/mg dL(-1)/cm(2)) and rapid detection (20 s). This proposed strategy of biomolecule detection is thus a promising platform for the development of miniaturized device for biosensing applications.

Lipid–Lipid Interactions in Aminated Reduced Graphene Oxide Interface for Biosensing Application

A label-free biosensor based on antiapolipoprotein B 100 functionalized-aminated reduced graphene oxide interface has been fabricated for detection of low density lipoprotein (LDL or lipid) cholesterol. The aminated reduced graphene oxide (NH2-rGO) based electrode surface is covalently functionalized with antiapolipoprotein B 100 (AAB or lipid) using EDC/NHS coupling chemistry. The lipid-lipid interactions at the NH2-rGO electrode surface have been investigated using electrochemical impedance spectroscopic technique. The structural and morphological investigations of NH2-rGO based immunosensor have been accomplished via transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, UV-visible, and electrochemical techniques. The impedimetric response of the proposed immunosensor shows excellent sensitivity (612 Ω mg(-1) dL cm(-2)), a response time of 250 s, and a low detection limit of 5 mg/dL of LDL molecules. The association, dissociation, and equilibrium rate constants for this immunoelectrode are found to be 1.66 M(-1) s(-1), 0.6 s(-1), and 2.77 M(-1), respectively. The long-term stability and excellent reproducibility of the proposed immunosensor indicates a suitable platform for detection of LDL or lipid molecules. This immunosensor provides an efficient platform for analysis of the antigen-antibody interactions of lipid molecules.

Highly Efficient Bienzyme Functionalized Nanocomposite-Based Microfluidics Biosensor Platform for Biomedical Application

This report describes the fabrication of a novel microfluidics nanobiochip based on a composite comprising of nickel oxide nanoparticles (nNiO) and multiwalled carbon nanotubes (MWCNTs), as well as the chips use in a biomedical application. This nanocomposite was integrated with polydimethylsiloxane (PDMS) microchannels, which were constructed using the photolithographic technique. A structural and morphological characterization of the fabricated microfluidics chip, which was functionalized with a bienzyme containing cholesterol oxidase (ChOx) and cholesterol esterase (ChEt), was accomplished using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy. The XPS studies revealed that 9.3% of the carboxyl (COOH) groups present in the nNiO-MWCNT composite are used to form amide bonds with the NH2 groups of the bienzyme. The response studies on this nanobiochip reveal good reproducibility and selectivity, and a high sensitivity of 2.2 mA/mM/cm2. This integrated microfluidics biochip provides a promising low-cost platform for the rapid detection of biomolecules using minute samples.

Mediator-free microfluidics biosensor based on titania–zirconia nanocomposite for urea detection

Urease (Urs) and glutamate dehydrogenase (GLDH) co-immobilized onto titania–zirconia (TiO2–ZrO2) nanocomposite and integrated with microfluidics mediator-free sensor have been utilized for urea detection. The PDMS microchannels have been sealed with a glass substrate comprising of reference (Ag/AgCl), counter (ITO) and working (Urs-GLDH/TiO2-ZrO2/ITO) electrodes. This mediator-free microfluidics urea sensor shows linearity as 5–100 mg/dL with improved sensitivity as 2.74 μA [Log mM]−1 cm−2 and detection limit of 0.07 mg/dl (0.44 mM) using 3σb/m criteria. The Reynolds number has been found to be as 0.166, indicating that fluid flow is completely laminar, controllable and the pressure drop across the microchannels is found to be as 3.5 × 103 Pa.

Microfluidic Immuno-Biochip for Detection of Breast Cancer Biomarkers Using Hierarchical Composite of Porous Graphene and Titanium Dioxide Nanofibers

We report on a label-free microfluidic immunosensor with femtomolar sensitivity and high selectivity for early detection of epidermal growth factor receptor 2 (EGFR2 or ErbB2) proteins. This sensor utilizes a uniquely structured immunoelectrode made of porous hierarchical graphene foam (GF) modified with electrospun carbon-doped titanium dioxide nanofibers (nTiO2) as an electrochemical working electrode. Due to excellent biocompatibility, intrinsic surface defects, high reaction kinetics, and good stability for proteins, anatase nTiO2 are ideal for electrochemical sensor applications. The three-dimensional and porous features of GF allow nTiO2 to penetrate and attach to the surface of the GF by physical adsorption. Combining GF with functional nTiO2 yields high charge transfer resistance, large surface area, and porous access to the sensing surface by the analyte, resulting in new possibilities for the development of electrochemical immunosensors. Here, the enabling of EDC-NHS chemistry covalently immobilized the antibody of ErbB2 (anti-ErbB2) on the GF-nTiO2 composite. To obtain a compact sensor architecture, the composite working electrode was designed to hang above the gold counter electrode in a microfluidic channel. The sensor underwent differential pulse voltammetry and electrochemical impedance spectroscopy to quantify breast cancer biomarkers. The two methods had high sensitivities of 0.585 μA μM(-1) cm(-2) and 43.7 kΩ μM(-1) cm(-2) in a wide concentration range of target ErbB2 antigen from 1 × 10(-15) M (1.0 fM) to 0.1 × 10(-6) M (0.1 μM) and from 1 × 10(-13) M (0.1 pM) to 0.1 × 10(-6) M (0.1 μM), respectively. Utilization of the specific recognition element, i.e., anti-ErbB2, results in high specificity, even in the presence of identical members of the EGFR family of receptor tyrosine kinases, such as ErbB3 and ErbB4. Many promising applications in the field of electrochemical detection of chemical and biological species will derive from the integration of the porous GF-nTiO2 composite into microfluidic devices.

Nanomaterial-based biosensors for food toxin detection.

There is an increased interest toward the development of bioelectronic devices for food toxin (mycotoxins) detection. Mycotoxins are highly toxic secondary metabolites produced by fungi like Fusarium, Aspergillus, and Penicillium that are frequently found in crops or during storage of food including cereals, nuts, fruits, etc. The contamination of food by mycotoxins has become a matter of increasing concern. High levels of mycotoxins in the diet can cause adverse, acute, and chronic effects on human health and a variety of animal species. Side effects may particularly affect the liver, kidney, nervous system, endocrine system, and immune system. Among 300 mycotoxins known till date, there are a few that are considered to play an important part in food safety, and for these, a range of analytical methods have been developed. Some of the important mycotoxins include aflatoxins, ochratoxins, fumonisins, citreoviridin, patulin, citrinin, and zearalenon. The conventional methods of analysis of mycotoxins normally require sophisticated instrumentation, e.g., liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. Hence, new analysis tools are necessary to attain more sensitive, specific, rapid, and reliable information about the desired toxin. For the last about two decades, the research and development of simpler and faster analytical procedures based on affinity biosensors has aroused much interest due to their simplicity and sensitivity. The nanomaterials have recently had a great impact on the development of biosensors. The functionalized nanomaterials are used as catalytic tools, immobilization platforms, or as optical or electroactive labels to improve the biosensing performance to obtain higher sensitivity, stability, and selectivity. Nanomaterials, such as carbon nanomaterials (carbon nanotubes and graphene), metal nanoparticles, nanowires, nanocomposites, and nanostructured metal oxide nanoparticles are playing an increasing role in the design of sensing and biosensing systems for mycotoxin determination. Furthermore, these nanobiosystems are also bringing advantages in terms of the design of novel food toxin detection strategies. We will focus on some of the recent results related to fabrication of nanomaterial-based biosensors for food toxin detection obtained in our laboratories.

Nanostructured anatase-titanium dioxide based platform for application to microfluidics cholesterol biosensor

We report results of studies relating to the fabrication of a microfluidics cholesterol sensor based on nanocrystalline anatase-titanium dioxide (ant-TiO2) film deposited onto indium tin oxide (ITO) glass. The results of response studies (optimized under the flow rate of 30 μl/min) conducted on cholesterol oxidase (ChOx) immobilized onto crystalline ant-TiO2 nanoparticles (∼27 nm)/ITO microfluidics electrode reveal linearity as 1.3 to 10.3 mM and improved sensitivity of 94.65 μA/mM/cm2. The observed low value of Km (0.14 mM) indicates high affinity of ChOx to cholesterol. No significant changes in current response of this microfluidics sensor are measured in the presence of different interferents.

Tyrosinase conjugated reduced graphene oxide based biointerface for bisphenol A sensor

We have fabricated a nanocomposite of reduced graphene oxide (rGO) sheets and chitosan (Cn) polymer based highly sensitive electrochemical biosensor for detection of bisphenol A (BPA). The two-dimensional structure and chemical functionality of rGO and Cn provide an excellent electrode surface for loading of tyrosinase enzyme molecules. This rGO-Cn nanocomposite is capable of effectively utilizing their superior conductivity, larger effective surface area and superior electrochemical performance due to its synergistic effect between rGO and Cn. The structural, morphological and electrochemical characterizations of nanocomposite sheets have been performed by electron microscopy, X-ray diffraction, FTIR and Potentiostat/Galvanostat techniques. This fabricated biosensor is sensitive to nanomolar (0.74 nM) concentration of BPA and detection time is 10s compared to conventional BPA ELISA kit (0.3 µg/L and 2.5h). The rGO-Cn based biosensor exhibits a higher sensitivity (83.3 µA nM(-1) cm(-2)), wider linearity (0.01-50 µM) with good selectivity towards BPA. This biosensor is capable to quantify real sample of BPA using packaged drinking water bottles. This rGO-Cn nanocomposite sheets emerges as a potential electrode material for detection of other estrogenic substrate.

A self assembled monolayer based microfluidic sensor for urea detection

Urease (Urs) and glutamate dehydrogenase (GLDH) have been covalently co-immobilized onto a self-assembled monolayer (SAM) comprising of 10-carboxy-1-decanthiol (CDT) via EDC-NHS chemistry deposited onto one of the two patterned gold (Au) electrodes for estimation of urea using poly(dimethylsiloxane) based microfluidic channels (2 cm × 200 μm × 200 μm). The CDT/Au and Urs-GLDH/CDT/Au electrodes have been characterized using Fourier transform infrared (FTIR) spectroscopy, contact angle (CA), atomic force microscopy (AFM) and electrochemical cyclic voltammetry (CV) techniques. The electrochemical response measurement of a Urs-GLDH/CDT/Au bioelectrode obtained as a function of urea concentration using CV yield linearity as 10 to 100 mg dl(-1), detection limit as 9 mg dl(-1) and high sensitivity as 7.5 μA mM(-1) cm(-2).