Ved Varun Agrawal

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.

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.

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.

Protein-conjugated quantum dots interface: binding kinetics and label-free lipid detection.

We propose a label-free biosensor platform to investigate the binding kinetics using antigen-antibody interaction via electrochemical and surface plasmon resonance (SPR) techniques. The L-cysteine in situ capped cadmium sulfide (CdS; size < 7 nm) quantum dots (QDs) self-assembled on gold (Au) coated glass electrode have been covalently functionalized with apolipoprotein B-100 antibodies (AAB). This protein conjugated QDs-based electrode (AAB/CysCdS/Au) has been used to detect lipid (low density lipoprotein, LDL) biomolecules. The electrochemical impedimetric response of the AAB/CysCdS/Au biosensor shows higher sensitivity (32.8 kΩ μM(-1)/cm(2)) in the detection range, 5-120 mg/dL. Besides this, efforts have been made to investigate the kinetics of antigen-antibody interactions at the CysCdS surface. The label-free SPR response of AAB/CysCdS/Au biosensor exhibits highly specific interaction to protein (LDL) with association constant of 33.4 kM(-1) s(-1) indicating higher affinity toward LDL biomolecules and a dissociation constant of 0.896 ms(-1). The results of these studies prove the efficacy of the CysCdS-Au platform as a high throughput compact biosensing device for investigating biomolecular interactions.

Biocompatible nanostructured magnesium oxide-chitosan platform for genosensing application

A novel organic-inorganic platform comprising of chitosan (CH) modified nanostructured magnesium oxide (nanoMgO) has been electrophoretically deposited on the indium-tin-oxide (ITO) substrate. The single stranded probe DNA (ssDNA) sequence of Vibrio cholerae has been covalently functionalized onto CH-nanoMgO/ITO surface. The cytotoxicity assay of nanoMgO particles, examined using human intestinal cell line (INT 407), reveals no significant cytotoxicity at the given doses in the range of 50-350 μg/mL. The X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and various microscopic techniques have been employed for the structural and morphological analysis of the fabricated electrodes. The electrochemical response studies of ssDNA and fragmented genomic DNA hybridized electrode (dsGDNA/CH-nanoMgO/ITO) have been carried out using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The dsGDNA/CH-nanoMgO/ITO bioelectrode exhibits a linear response in the range 100-500 ng/μL with improved sensitivity of 36.72 nA/ng/cm(2), faster response time of 3s and high stability of 3-4 months under refrigerated condition. The lower detection limit of fabricated genosensor has been estimated as 35.20 ng/μL and it shows good reproducibility/repeatability.

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.

Protein Functionalized Carbon Nanotubes-based Smart Lab-on-a-Chip

A label-free impedimetric lab on a chip (iLOC) is fabricated using protein (bovine serum albumin) and antiapolipoprotein B functionalized carbon nanotubes-nickel oxide (CNT-NiO) nanocomposite for low-density lipoprotein (LDL) detection. The antiapolipoprotein B (AAB) functionalized CNT-NiO microfluidic electrode is assembled with polydimethylsiloxane rectangular microchannels (cross section: 100 × 100 μm). Cytotoxicity of the synthesized CNTs, NiO nanoparticles, and CNT-NiO nanocomposite has been investigated in the presence of lung epithelial cancer A549 cell line using MTT assay. The CNT-NiO nanocomposite shows higher cell viability at a concentration of 6.5 μg/mL compared to those using individual CNTs. The cell viability and proliferation studies reveal that the toxicity increases with increasing CNTs concentration. The X-ray photoelectron spectroscopy studies have been used to quantify the functional groups present on the CNT-NiO electrode surface before and after proteins functionalization. The binding kinetic and electrochemical activities of CNT-NiO based iLOC have been conducted using chronocoulometry and impedance spectroscopic techniques. This iLOC shows excellent sensitivity of 5.37 kΩ (mg/dL)(-1) and a low detection limit of 0.63 mg/dL in a wide concentration range (5-120 mg/dL) of LDL. The binding kinetics of antigen-antibody interaction of LDL molecules reveal a high association rate constant (8.13 M(-1) s(-1)). Thus, this smart nanocomposite (CNT-NiO) based iLOC has improved stability and reproducibility and has implications toward in vivo diagnostics.

Large Area Fabrication of Semiconducting Phosphorene by Langmuir-Blodgett Assembly

Phosphorene is a recently new member of the family of two dimensional (2D) inorganic materials. Besides its synthesis it is of utmost importance to deposit this material as thin film in a way that represents a general applicability for 2D materials. Although a considerable number of solvent based methodologies have been developed for exfoliating black phosphorus, so far there are no reports on controlled organization of these exfoliated nanosheets on substrates. Here, for the first time to the best of our knowledge, a mixture of N-methyl-2-pyrrolidone and deoxygenated water is employed as a subphase in Langmuir-Blodgett trough for assembling the nanosheets followed by their deposition on substrates and studied its field-effect transistor characteristics. Electron microscopy reveals the presence of densely aligned, crystalline, ultra-thin sheets of pristine phosphorene having lateral dimensions larger than hundred of microns. Furthermore, these assembled nanosheets retain their electronic properties and show a high current modulation of 104 at room temperature in field-effect transistor devices. The proposed technique provides semiconducting phosphorene thin films that are amenable for large area applications.