APTAMER BASED BIOSENSORS

Over the last decade there has been a continuous strive to replace antibodies and other labile biorecognition elements with stable and low-cost recognition systems for developing various diagnostic and detection devices. Among them, nucleic acid-based aptamers are emerging as efficient and viable alternatives to the antibodies. Our laboratory focusses on development of ssDNA aptamers against different biomarkers with an aim of developing aptasensors for diagnosis of malaria and myocardial infarction (MI) in point of care (PoC) and resource-limited environments. We have developed novel aptamers against FABP3 (a biomarker for MI), PfLDH, HRP-II, and PfGDH (all these three are malaria biomarkers) following the systematic evolution of ligand exponential enrichment (SELEX) procedure. SELEX involves screening of best ssDNA aptamer from a very large oligonucleotide library pool (10¹²-10¹⁵ molecules) against the target by iterative procedure of in vitro selection and enrichment process.

The enriched aptamer candidates were cloned, sequenced, screened and finally characterized with the help of different molecular and biophysical techniques to elucidate their 2-D and 3-D structures and target selectivity. We also study the specific interactions between the aptamer with the target to understand the specificity of the developed aptamers. In a study conducted during recent past, we explored combinatorial method to retrieve the possible specific binding modes and interaction patterns involved in large aptamer-protein complexes. Thus, the method we developed can be exploited to identify the optimum aptamer length for in-depth structure-function studies and its tailored applications.

The specific aptamers have been used to developed proof-of-concept for detection of PLDH, HRP-II, HFABP3, and PfGDH following optical (fluorescence, colorimetric, and pixel based) and electrochemical (impedance, capacitance, FET) transduction principles. One of the recent works is highlighted below. Here, a novel approach for detection of pan specific and Pf specific malaria has been developed by using a chromogenic reaction catalyzed by the corresponding biomarkers PLDH and PfGDH. We demonstrated that the approach could be implemented both in instrument-based laboratory settings and in instrument-free, paper-based, portable platforms. Using a specific aptamer coated over magnetic beads, the biomarkers could be captured and separated from the blood serum to perform the reaction. This strategy greatly excluded the potential interferences usually caused by the complex milieu of blood serum. The color developed on the modified paper may be used either for qualitative detection following a yes/no format through visual readout or for quantitative detection following color pixel-based response with an aid of camera integrated suitable software. Flexible detection capability of pan specific and Pf specific malaria, low cost, and interference-free detections of the biomarker enzymes from serum samples are the major advantages offered by the developed approaches.

BIOFUEL CELLS FOR POWER GENERATION AND BIOSENSOR APPLICATION

Biofuel cells (BFCs) are variants of chemical fuel cell where biological catalysts such as, redox enzymes and microorganisms, act as catalysts over the cell electrodes. Considering the operational criteria (room temperature and around physiological pH), type of catalysts (non-corrosive, biological sources), and fuels (amenable with renewable type) BFCs are regarded as green energy technology. Our current focus is the (1) alcohol fuel based enzymatic BFC with an aim of developing both alcohol sensor as well as energy generating device for powering small scale electronic devices and (2) cyanobacteria based BFCs for biosensing applications and waste water treatment for degradation of toxic aromatic organic compounds. In all the cases our effort is to develop proper electrical communication between the biocatalysts and the electrodes, for which we pursued direct electron transfer (DET) as the governing principle to harvest the biological electrons for generating power in the BFCs. To establish the DET, we explored various advance nanomaterials (e.g. MWCNTs, graphene nanoplatlets, magnetic nanoparticles etc) and conducting polymers for the fabrication of the bioelectrodes for BFCs. In a previous work, we reported an enzymatic BFC fabricated by alcohol oxidase (AOx) based bioanode for generating power from methanol substrate using air breathed laccase biocathode. The BFC generated an open circuit potential of 0.61V with maximum power density of 46 (+/- 0.002) μWcm^-2 at an optimum of 1M methanol concentration (Biosensors and Bioelectronics 59,184-191, 2014).

The interest on cyanobacteria as BFC catalysts is sharply increasing owing to many advantages being identified on their use in the BFCs. These photo-synthetic microorganisms are widespread in nature and can grow heterotrophically as well as photo-autotrophically in a self-sustainable manner. The application potential of these microorganisms for combined power generation and waste treatment through photosynthetic microbial fuelcell (PMFC) technology is vast due to their inherent survival capacity at high salt concentrations, in the presence of organic contaminants and under adverse environmental conditions. To implement the concept of DET in the PMFC a naturally sustained close contact between the bacterial cells and the conductive electrode is a desired condition. To meet this condition, the creation of a natural bacterial biofilm over the electrode surface in a short span of time would be a prudent approach for generating stable current from the cellular electrons in the fuel cell setup. We developed a novel silk-based nanocomposite matrix endowed with biocompatible, optoelectronic and electroactive properties suitable for bioanode fabrication for a PMFC by rationally doping QD and GNP in the silk-fibroin. The nanocomposite matrix promoted rapid biofilm growth of Synechococcus sp., supported FRET to surge the photosystems of the underneath bacteria in the biofilm and provided an electroactive surface for relaying metabolic electrons from the cells to the electrode through DET during operation of the PMFC. The cumulative action of these properties not only enhanced the power density, but also stabilized the power during the dark phase of the PMFC operation. We envision that our approach will be a big step forward, not only to improve the overall current density in a PMFC, but also to sustain its power at low light operating conditions, due to FRET guided surging of the photosystems of the cyanobacteria.

We have developed a new signal form for detection of alcohol following the cyanobacteria based PMFC. The proof-of-concept was first validated in a lab-scale PMFC and latter, it was translated into a paper based PMFC (p-PMFC). The p-PMFC relies on a novel signal response of alcohol mediated membrane disruption-linked potential burst. On interaction with injected alcohol, the degradation of the cell membrane increases the exposure of the electron transfer proteins to the anode leading to the instantaneous potential burst. This miniaturized (25 cm²), self-powered, disposable sensor exhibited a response time of ~10 s with a detection limit of 0.02 % for alcohol. Ethanol, owing to its lower polarity than has a greater effect on the fluidity and permeability of the membrane induces higher stress in cyanobacteria at a shorter time scale as compared to methanol. The p-PMFC device showed absolute selectivity towards ethanol at concentration < 0.05 %.

We also demonstrated for the first time the high catalytic potential of the cyanobacterial strain, Synechococcus sp. for toxic azo dye degradation (89 %), dye decolorization (> 68.5 %) and simultaneous power generation (4.9 +/- 0.5 Wm-3) in a PMFC. Herein, the nanofabricated TCP (Torrey carbon paper) anode developed by using polymer (PANI-co-PPy) coated MNPs(Magnetic nanoparticles) has offered multiple functions facilitating to enhance the power density in the PMFC and concomitant substantial degradation of the dye. Among the advantages offered by the anode, support for high biofilm growth that generated large amount of catalytic cells, increase interaction between the cells and the base electrode (due to high magnetic force) that facilitated metabolic electron flux to the electrode, and activating the cyanobacteria cells for producing high ROS with expected dual functions of dye degradation and charge transfer on the electrodes are prominent. Overall, this study validated that the cyanobacteria based PMFC with MNPs fabricated anode as potential technological solution for combined power generation and azo-dye degradation cum decolorization of industrial wastewater.

DEVELOPMENT OF ENZYME BASED RECOGNITION SYSTEM AND BIOSENSING PLATFORMS

Enzyme based biosensor for alcohol, cholesterol, and bilirubin is one of the major research activities in our lab. Sensitive signal transduction, high operational stability and extended shelf-life are the central issues being addressed to develop efficient biosensor for these targets. Different advance nanomaterial and nano crystals were studied to generate and amplify the signal under different configurations with these protein based catalysts following DET as governing principle. Some of the major achievements during recent past on the line are as follows: Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin (Bioelectrochemistry, 111, 7-14, 2016) and also their applications as fluorometric and colorimetric probe for detection of bilirubin (Biosensors and Bioelectronics, 59, 370-376, 2014). Alcohol oxidase protein mediated in-situ synthesized and stabilized gold nanoparticles for developing amperometric alcohol biosensor (Biosensors and Bioelectronics, 69,155-161, 2015). We reported silk Mat as a biocompatible-matrix for the immobilization of cholesterol oxidase (ChOx) to develop stable cholesterol biosensors. Further, a novel immobilized technique for ChOx through self-assembled process on gold nanoparticles was developed for sensing cholesterol following amperometric principle (Biosensors and Bioelectronics 26,3037-3043, 2011).The ultimate aim of all our sensor works (including aptamer, BFC and enzyme and nanozymes) is to develop portable device for application in point of care (PoC) and resource limited environments. Small sample volume, rapid analysis, reliable and low cost is the other criteria for developing biosensors of practical use. Low cost, lightweight and biocompatible materials integrating to microfluidic technology are being explored for such devices. Microfluidic paper-based sensor surface is one of our interests that have been pursued in our lab.