NANO-ENABLED BIOSENSORS FOR EFFICIENT DETECTION OF CANCER BIOMARKERS

Abstract

In the current scenario, the dominancy of cancer is becoming a disastrous threat to the mankind. Currently, it is the second most death-causing disease accounting for around 10 million death and 19.3 million new cancer cases in 2020. Therefore, an advanced analytical approach is desired as the need of the hour for the early diagnosis to curb the menace of cancer and to provide an efficient therapeutic response resulting in lesser morbidity and a higher survival rate. In line with this, the detection of cancer-specific biomarkers with higher sensitivity and accuracy has been reported as an efficient remedy for the early diagnosis, monitoring disease progression, designing the response to chemotherapeutic treatment, risk assessment, and accurate pre-treatment staging. Among different cancer biomarkers, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) stand out most widely used biomarkers for early cancer detection. There is a substantial difference in the serum concentration of these biomarkers in healthy beings and cancer patients. For example, in healthy human beings, the serum CEA concentration reaches up to 0-2.5 ng mL−1 and a higher level of 2-5 ng mL−1 in smokers. However, elevated serum CEA concentration (≥20 ng mL−1) in patients greatly indicates the presence of cancer. Similarly, studies have reported that neuron-specific enolase (NSE) is a reliable, specific, and sensitive serum biomarker for the early diagnosis of small cell lung cancer (SCLC) and could assess the patient's recovery progress. In normal human beings, the serum NSE concentration has been recorded as up to 12-13 ng mL−1, whereas, in SCLC patients, serum NSE concentration is found to be more than 35 ng mL−1. Recently, non-radiative energy transfer-based fluorescent biosensors have been found to possess remarkable potential for simple, fast, and reliable analyte detection owing to their various advantages such as the requirement of simple instrumentation, reduced costs of testing, low power consumption, the capability of real-time detection, multi-analyte detection, easy operation, and highly sensitive response with minimum background noise. It has been reported that the selection of donor-acceptor material is vital for improving the efficiency and performance of these biosensors. In line with this, graphene quantum dots (GQDs) have gradually evolved as a superior alternative to traditional fluorophores and energy donor species such as organic dyes, semiconductors, and toxic heavy metal-based quantum dots, etc., owing to their remarkable optoelectronic properties. GQDs endow distinct properties such as strong quantum confinement and edge effects, presence of several active functional groups, higher aqueous solubility, tunable bandgap, broad excitation spectra, and good photostability. Despite numerous interesting properties, the short fluorescence lifetime and relatively low quantum yield (QY) of GQDs hamper their commercial aspects in developing different biosensing platforms. On the other hand, two-dimensional (2D) and zero-dimensional (0D) nanomaterials, including graphene derivatives (reduced graphene oxide (rGO), graphene oxide (GO)), Ti3C2-MXene and metal nanoparticles (gold and silver nanoparticles (AuNPs and AgNPs) have attained considerable attention as proficient energy acceptor species owing to their ability to accept transferred energy even at a longer distance following the nano surface energy transfer (NSET) phenomenon. Besides, nanohybrids of 0D and 2D nanomaterials comprising graphene derivatives, metal nanoparticles, and Ti3C2-MXene have opened new avenues in fabricating highly efficient fluorescent biosensors. In the present thesis, attempts have been made toward fabricating selective, rapid, label-free, and highly sensitive fluorescent biosensing platforms for quantitative detection of cancer biomarkers. The functionality of the fabricated biosensors depends on the energy transfer (fluorescence quenching) from the donor species to the nearby acceptor species, followed by the gradual recovery of quenched fluorescence after specific biomarker addition. The amount of recovered fluorescence depends on the cancer biomarker concentration in the sample, thus providing the biomarker's quantitative determination. The specific antigen-antibody interactions ensure the specificity of the fabricated fluorescent biosensing platforms. In Chapters 1-3, the introduction, literature review, and experimental procedures have been elucidated. In Chapter 4, the effect of heteroatom doping, and surface functionalization has been investigated on the optical properties of GQDs. The in-situ nitrogen-doped and amine functionalized GQDs (amine-N-GQDs) are synthesized employing a one-pot and the bottom-up hydrothermal method. In Chapter 5, the obtained amine-N-GQDs are protein functionalized with NSE-specific monoclonal antibodies (anti-NSE) using the standard EDC-NHS chemistry. The biofunctionalized GQDs (anti-NSE/amine-N-GQDs) and AuNPs are utilized as the donor acceptor pair to fabricate the fluorescent biosensing platform for the quantitative NSE detection in standard and spiked serum samples. In Chapter 6, a nanocomposite of AuNPs and rGO (AuNPs@rGO) has been synthesized following the one-pot in-situ low-temperature reduction method. Besides, the earlier synthesized amine-N-GQDs are protein functionalized with anti-CEA antibodies to utilize them as donor species. The efficient energy transfer kinetics have been envisaged using the AuNPs@rGO nanocomposite as a dual quencher compared to AuNPs as a single quencher. Further, a potential biosensing platform based on anti-CEA/amine-N-GQDs/AuNPs@rGO immunoprobe has been developed for quantitative CEA detection. In chapter 7, facile synthetic regimes have been worked upon to synthesize bare Ti3C2-MXene and Ag@Ti3C2-MXene nanohybrid. The Ti3C2-MXene nanosheets are decorated with AgNPs to obsolete the agglomeration and restacking through a one-pot direct reduction method wherein the Ti3C2-MXene nanosheets act both as a reducing agent and support matrix for AgNPs. The formulated Ag@Ti3C2-MXene nanohybrid has been utilized as the dual energy acceptor in a single system. The quenching efficiency and energy transfer capability between the anti NSE/amino-GQDs (donor) and Ag@Ti3C2-MXene (acceptor) have been explored through steady-state and time-resolved spectroscopic studies and compared with bare Ti3C2-MXene, AgNPs, graphene, and AuNPs. Based on optimized donor-acceptor pair, a rapid, label-free, and highly sensitive fluorescent turn-on biosensing system is constructed for quantitative and sensitive NSE detection. Finally, the biosensing performance of Ag@Ti3C2-MXene nanohybrid based platform is compared with bare Ti3C2-MXene, and graphene-based platforms for NSE detection.