Engineering of nano-bio interfaces towards the development of portable biosensors

The rapid development in bio-nanotechnology coupled with advancements in microelectronics and computing power has given rise to an enormous potential for the development of point-of-care diagnostic devices and portable biosensors. The application area encompasses human disease detection, analyzing bio-hazardous molecules and toxins and other inorganic materials. Amongst the plethora of receptors in such biosensors, aptamers, which are oligonucleotides with high affinity for target analytes, have demonstrated considerably improved performance over traditional receptors like antibodies. The hallmark of a portable biosensor is high throughput in response to analyte characterized by appreciable signal/noise ratio, dynamic range and low limit of detection. However, these characteristics are difficult to achieve in practical scenarios due to a variety of factors and thus the success is mostly limited to lab based developments. Particularly, the poor commercial success of portable biosensors directed towards human disease detection is noticeable. In this study, effort has been made to address some of the issues like external control over target binding to receptor, low signal/noise ratio and detection in presence of interfering molecules, challenging the practical deployment of a portable aptamer-based biosensor by focusing on engineering of the nano-bio interface of the transducer in such devices. The investigation is carried out in three directions: 1. Electrical actuation of receptor-target complex 2. Impedance characterization of nanoporous transducer 3. Engineering of the receptor arrangement on the transducer The goal of the present research is to provide improved understanding of such processes which can lead to the design and creation of a point-of-care diagnostic device with the ability to detect human disease markers like: (i) Ebola virus protein sGP/GP1-GP2 as a biomarker for Ebola virus infection (ii) NGAL protein related to Acute Kidney Injury Through the research described in this thesis, the following have been understood: (a) The actuation of aptamer-protein complex, where the protein may be dissociated from the aptamer immobilized on the electrode surface due to externally applied electric field depends on the length of the aptamer nucleotide sequence, charge of protein and the surface grafting density of aptamer. (b) Four-electrode electrochemical sensors, utilizing nanoporous alumina membrane with aptamer immobilized on one surface can be used as a biosensor in which membrane impedance depends on target concentration and greater sensitivity is observed with serum albumin in the sample to be tested. Also, there exists an optimal frequency for aptamer-protein complexes which can help in reducing the time of operation of such a biosensor by eliminating the need for long range of frequency scans to get system impedance. (c) Competition mode of sensing, using a receptor weakly attached to the surface via a linker, and having higher propensity to bind with target analyte in the solution compared to remaining surface bound, may be used as a technique to increase signal/noise ratio by increasing sensitivity to low concentration analyte in presence of interfering molecules

Application of functionalized graphene oxide based biosensors for health monitoring: Simple graphene derivatives to 3D printed platforms

Biosensors hold great potential for revolutionizing personalized medicine and environmental monitoring. Their construction is the key factor which depends on either manufacturing techniques or robust sensing materials to improve efficacy of the device. Functional graphene is an attractive choice for transducing material due to its various advantages in interfacing with biorecognition elements. Graphene and its derivatives such as graphene oxide (GO) are thus being used extensively for biosensors for monitoring of diseases. In addition, graphene can be patterned to a variety of structures and is incorporated into biosensor devices such as microfluidic devices and electrochemical and plasmonic sensors. Among biosensing materials, GO is gaining much attention due to its easy synthesis process and patternable features, high functionality, and high electron transfer properties with a large surface area leading to sensitive point-of-use applications. Considering demand and recent challenges, this perspective review is an attempt to describe state-of-the-art biosensors based on functional graphene. Special emphasis is given to elucidating the mechanism of sensing while discussing different applications. Further, we describe the future prospects of functional GO-based biosensors for health care and environmental monitoring with a focus on additive manufacturing such as 3D printing

Characterization of the influence of external stimulus on protein-nucleic acid complex through multiscale computations

The concomitant detection, monitoring and analysis of biomolecules have assumed utmost importance in the field of medical diagnostics as well as in different spheres of biotechnology research such as drug development, environmental hazard detection and biodefense. There is an increased demand for the modulation of the biological response for such detection / sensing schemes which will be facilitated by the sensitive and controllable transmission of external stimuli. Electrostatic actuation for the controlled release/capture of biomolecules through conformational transformations of bioreceptors provides an efficient and feasible mechanism to modulate biological response. In addition, electrostatic actuation mechanism has the advantage of allowing massively parallel schemes and measurement capabilities that could ultimately be essential for biomedical applications.Experiments have previously demonstrated the unbinding of thrombin from its aptamer in presence of small positive electrode potential whereas the complex remained associated in presence of small negative potentials / zero potential. However, the nanoscale physics/chemistry involved in this process is not clearly understood. In this thesis a combination of continuum mechanics based modeling and a variety of atomistic simulation techniques have been utilized to corroborate the aforementioned experimental observations. It is found that the computational approach can satisfactorily predict the dynamics of the electrically excited aptamer-thrombin complex as well as provide an analytical model to characterize the forced binding of the complex.</p

Application of Functionalized Graphene Oxide Based Biosensors for Health Monitoring: Simple Graphene Derivatives to 3D Printed Platforms

Biosensors hold great potential for revolutionizing personalized medicine and environmental monitoring. Their construction is the key factor which depends on either manufacturing techniques or robust sensing materials to improve efficacy of the device. Functional graphene is an attractive choice for transducing material due to its various advantages in interfacing with biorecognition elements. Graphene and its derivatives such as graphene oxide (GO) are thus being used extensively for biosensors for monitoring of diseases. In addition, graphene can be patterned to a variety of structures and is incorporated into biosensor devices such as microfluidic devices and electrochemical and plasmonic sensors. Among biosensing materials, GO is gaining much attention due to its easy synthesis process and patternable features, high functionality, and high electron transfer properties with a large surface area leading to sensitive point-of-use applications. Considering demand and recent challenges, this perspective review is an attempt to describe state-of-the-art biosensors based on functional graphene. Special emphasis is given to elucidating the mechanism of sensing while discussing different applications. Further, we describe the future prospects of functional GO-based biosensors for health care and environmental monitoring with a focus on additive manufacturing such as 3D printing

Engineering of nano-bio interfaces towards the development of portable biosensors

The rapid development in bio-nanotechnology coupled with advancements in microelectronics and computing power has given rise to an enormous potential for the development of point-of-care diagnostic devices and portable biosensors. The application area encompasses human disease detection, analyzing bio-hazardous molecules and toxins and other inorganic materials. Amongst the plethora of receptors in such biosensors, aptamers, which are oligonucleotides with high affinity for target analytes, have demonstrated considerably improved performance over traditional receptors like antibodies. The hallmark of a portable biosensor is high throughput in response to analyte characterized by appreciable signal/noise ratio, dynamic range and low limit of detection. However, these characteristics are difficult to achieve in practical scenarios due to a variety of factors and thus the success is mostly limited to lab based developments. Particularly, the poor commercial success of portable biosensors directed towards human disease detection is noticeable. In this study, effort has been made to address some of the issues like external control over target binding to receptor, low signal/noise ratio and detection in presence of interfering molecules, challenging the practical deployment of a portable aptamer-based biosensor by focusing on engineering of the nano-bio interface of the transducer in such devices. The investigation is carried out in three directions: 1. Electrical actuation of receptor-target complex 2. Impedance characterization of nanoporous transducer 3. Engineering of the receptor arrangement on the transducer The goal of the present research is to provide improved understanding of such processes which can lead to the design and creation of a point-of-care diagnostic device with the ability to detect human disease markers like: (i) Ebola virus protein sGP/GP1-GP2 as a biomarker for Ebola virus infection (ii) NGAL protein related to Acute Kidney Injury Through the research described in this thesis, the following have been understood: (a) The actuation of aptamer-protein complex, where the protein may be dissociated from the aptamer immobilized on the electrode surface due to externally applied electric field depends on the length of the aptamer nucleotide sequence, charge of protein and the surface grafting density of aptamer. (b) Four-electrode electrochemical sensors, utilizing nanoporous alumina membrane with aptamer immobilized on one surface can be used as a biosensor in which membrane impedance depends on target concentration and greater sensitivity is observed with serum albumin in the sample to be tested. Also, there exists an optimal frequency for aptamer-protein complexes which can help in reducing the time of operation of such a biosensor by eliminating the need for long range of frequency scans to get system impedance. (c) Competition mode of sensing, using a receptor weakly attached to the surface via a linker, and having higher propensity to bind with target analyte in the solution compared to remaining surface bound, may be used as a technique to increase signal/noise ratio by increasing sensitivity to low concentration analyte in presence of interfering molecules.</p

Electrical Stimulus Controlled Binding/Unbinding of Human Thrombin-Aptamer Complex

The binding/unbinding of the human thrombin and its 15-mer single stranded DNA aptamer, under the application of external stimulus in the form of electrostatic potential/electric field, is investigated by a combination of continuum analysis and atomistic molecular dynamics simulation. In agreement with the experiments that demonstrate the influence of electrostatic potential on the thrombin/aptamer complex, our computations show that the application of positive electric field successfully unbinds the thrombin from the aptamer. Results from umbrella sampling simulations reveal that there is a decrease in the free energy of binding between the thrombin and aptamer in presence of positive electric fields. Hydrogen bonding and non-bonded interaction energies, and hence the free energy of binding, between the thrombin and its aptamer reduce as the applied electric field is shifted from negative to positive values. Our analyses demonstrate that application of electrical stimulus modifies the molecular interactions within the complex and consequently, electrical field can be used to modulate the association between the thrombin and its aptamer.This article is published as Gosai, Agnivo, Xiao Ma, Ganesh Balasubramanian, and Pranav Shrotriya. "Electrical Stimulus Controlled Binding/Unbinding of Human Thrombin-Aptamer Complex." Scientific Reports 6 (2016): 37449. 10.1038/srep37449. Posted with permission.</p

Multiscale Modeling Reveals the Cause of Surface Stress Change on Microcantilevers Due to Alkanethiol SAM Adsorption

Experimental results show that the adsorption of the self assembled monolayers (SAMs) on a gold surface induces surface stress change that cause a deformation of the underlying substrate. However, the exact mechanism of stress development is yet to be elucidated. In the present study, multiscale computational models based on molecular dynamics (MD) simulations are applied to study the mechanism governing surface stress change. Distinct mechanisms for adsorption induced surface deformation, namely inter chain repulsion and thiol-gold interaction driven gold surface reconstruction, are investigated. Two different inter-atomic potentials, embedded atom method (EAM) and surface embedded atom method (SEAM), are used in the MD simulations to study the reconstruction induced surface stresses. Comparison of the predicted surface stress changes, resulting from MD and continuum mechanics based models, with observed experimental response, indicate that a modified SEAM based multiscale model can better capture the surface stress changes observed during alkanethiol SAM formation and gold surface reconstruction is the primary factor behind the surface stress change. Inter chain repulsions of SAM are found to have minimal contribution. Also, both the simulations and experiments show that surface stress change increases with surface coverage density and larger grain size.This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Journal of Chemical Information and Modeling, copyright © American Chemical Society after peer review. To access the final edited and published work see DOI: 10.1021/acs.jcim.0c00146. Posted with permission.</p

Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1,2 and Their Application in Electrochemical Sensing

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2′FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.This article is published as Banerjee, Soma, Mahsa Askary Hemmat, Shambhavi Shubham, Agnivo Gosai, Sivaranjani Devarakonda, Nianyu Jiang, Charith Geekiyanage et al. "Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1, 2 and Their Application in Electrochemical Sensing." International Journal of Molecular Sciences 24, no. 5 (2023): 4627. DOI: 10.3390/ijms24054627. Copyright 2023 by the authors. Attribution 4.0 International (CC BY 4.0). Posted with permission