Development of Liquid Crystals Based Biosensor for the Detection of HIV-1 Surface Glycoprotein-120

Abstract

HIV-1 (Human Immunodeficiency Virus-1) is the causative agent of the deadliest disease AIDS (Acquired Immunodeficiency Disorder Syndrome) with no cure and vaccine till now. Around 36.3 million died due to AIDS and AIDS-related illnesses since the beginning of this disorder. However, early diagnosis may turn this fatal disease into a manageable chronic disease leading to minimal death and economic loss. Since surface glycoprotein-120 (gp-120) of the HIV-1 virus plays an important role in HIV-1 infectivity, we chose, gp-120 as a target protein for HIV-1 early detection. However, it is very difficult to detect HIV-1 during the early stage of its infection. Conventional methods for HIV-1 detection are ELISA, Rapid test assays (e.g., oral home test and rapid antibody test), PCR, and P24 antigen assay. These methods are either laborious, complicated, or require additional tests for confirmation. Therefore, there is a dire need for a rapid and economical sensing system that can detect HIV-1 early and hence saves many lives by reducing the risk of virus transmission. Since liquid crystals (LCs) based bioassays have grown highly popular because of their labelfree nature, excellent sensitivity, and real-time monitoring capacity, we aimed our study to design LCs-based biosensing assays for HIV-1 detection. Furthermore, we have developed and proposed LCs-based biosensing assays by integrating LCs-based biosensing with other techniques such as microfluidics and microcontact printing. In this thesis, firstly, we presented an easy and label-free aptasensing method based on LCs for the detection of gp-120. The LCs were supported on the surface of a glass slide coated with DMOAP and then covered with arrays of drops with the suitable concentration of B40t77 aptamer. The right concentration of B40t77 aptamer enables homeotropic alignment of the LCs. Due to the disruption of LCs homeotropic alignm -ent caused by the contact between B40t77 RNA aptamer and HIV-1 surface gp-120, a notable topological change was seen on the surface. Dark-to-bright transition is the result, which was seen using a polarised optical microscope. In addition to enabling the identification of gp-120, this designed biosensing device also allowed quantitative analysis of the results using Image J software. The limit of detection of gp-120 was found to be 0.2 µg/ml. Regarding the platform’s selectivity, no response was observed when gp-120 was substituted with other proteins such as bovine serum albumin (BSA),

v hepatitis A virus capsid protein 1 (Hep A VP1), and immunoglobulin G protein (IgG). The given biosensing technology offers the possibility to create. The given biosensing technology offers the possibility to create a functional device for the fast detection of HIV-1 surface gp-120 due to properties like high specificity, no requirement for instrumental read-out, and label-free nature. Furthermore, B40t77 RNA aptamer-based binding bioassay was purposed, which recognizes and binds specifically to gp-120 and has also been investigated and reported. The proposed bioassay platform was built upon a DMOAP-modified hydrophobic glass slide with surface-immobilized gp-120 patterns by using a TEM gold grid as a mold. By exploiting PDMS stamp with microchannels to prepare a closed microfluidic device by mounting physically on a modified hydrophobic glass slide with gp-120 (target) patterns on the surface. When the solution containing different concentrations of B40t77 is allowed to flow through channels, the aptamer can interact with gp-120 specifically and remove it from the surface in a time of less than 30 min. The total assay time observed was 30 min. The outcomes of the binding event can be enhanced in the last phase by converting LCs into optical outputs. This bioassay can detect as low as 1 μg/ml of gp-120 with high specificity within 30 min. No response is obtained when other proteins like BSA were used instead of the target protein gp-120. This is the first qualitative biosensing platform that allows simple detection of gp-120 with the naked eye. The proposed bioassay is robust, cheap, and does not require additional labeling. Thus, this bioassay is potentially useful for the early detection of HIV-1 in resources-limited regions. Finally, an LC-based biosensing assay was developed by integrating LCs-based sensing and microcontact printing (µCP). A functional template made up of in situ synthesized gold nanoparticles (AuNPs) is prepared on polydimethylsiloxane (PDMS) for the patterning of target protein onto the desired solid substrates. Unlike the previous studies in which bioreceptor probes are randomly attached to the PDMS stamp through electrostatic interactions, herein, we proposed an AuNPs-PDMS stamp, which provides the surface for the attachment of thiol-modified biorecognition probes to link to the stamp surface through a dative bond with a single anchoring point based on thiol chemistry. By using this platform, we have proposed the ability of µCP to selectively capture and transfer target protein onto solid surfaces for detection purposes. At the

vi end of µCP, we have also investigated whether LCs could be used as a label-free approach for identifying transfer protein. Our reported approach provides a promise to exploit it for the biosensing of various analytes. The proposed LCs-based biosensing assays for HIV-1 gp-120 have the potential to contribute to AIDS diagnosis in the early stage