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Chapter 5: Phenylboronic Acid Templated Gold Nanoclusters for Mucin Detection Using a

5.1. Introduction



106 Chapter 5

boronic acids hold the potential to act as anticancer agents. However, for the application of boronic acids as anticancer agents, it is essential to track their distribution inside the cells, and this can be achieved via tagging of the therapeutic boron-based compounds with imaging probes. Rapid sensing methods based on specific targeting of sialic acid could be also useful for the development of point- of-care (POC) diagnostic devices and is of importance in targeted delivery and therapeutics. An important secretory glycoprotein, mucin, contains sialic acid moieties and is found to be associated with pathways in cancer progression, invasion, and metastasis. Also, mucins constitute a major portion of the tumor area/volume in many cases.(15) However, the rapid detection of mucin is challenging because current standard methods, like histochemical analysis of tumor tissues through PAS/Alcain blue staining,(16,17) are invasive and time-consuming and alternative approaches, such as enzyme-linked immunosorbent assays, antibody-mediated field-effect-transistor devices, and aptamer-based fluorometric assays,(18-22) require expensive antibody-mediated reactions and complicated labeling steps for signal generation. Thereby, the development of a rapid “one-step”

assay for analysis of mucins present in body fluids, tumor biopsy, etc., could enable integration to POC platforms for in vitro diagnostics. A key element would be its specific recognition using labeling molecules for adequate signal generation using minimal precursors in order to keep the operation of the device simple, accurate, and specific.

Therefore, an important factor to either use boronic acid template systems for the development of rapid in vitro POC assays to detect mucin or track intracellular distribution during therapeutic regime inside cells is to achieve effective tagging with reporter molecules. In this regard, fluorometric markers are promising because of their fast response and sensitivity and, hence, are widely used in the detection of biomolecular analytes and bioimaging. Although fluorescence labeling is a daunting challenge with respect to small molecules like phenylboronic acid, it is mostly favored and considered to be advantageous because of its applications in theranostics.

Among fluorescence reporters, organic dyes had been a choice for decades in fluorescence-based studies, but their applications in biological experiments are limited because of their low photostability, toxicity (in certain cases), and poor solubility in water.(23) Nanomaterials like quantum dots, C dots, etc., although a viable option, often require either high-temperature conditions or purification steps and take a long time, and related toxicity concerns arising from the use of heavy metals in the synthesis of quantum dots hinder their extensive usage.(24-27) Recently, noble-metal nanoclusters of gold, silver, and copper have emerged as promising candidates for sensing assays and biological applications because of their biocompatibility, photostability, and large Stokes shift.(28) However, the majority of available techniques involve the presynthesis of nanomaterials and then require functionalization to achieve effective conjugation with receptors for recognition purposes. This not only introduces an extra step but also increases the possibility of the loss of properties of the nanomaterial or the biological component involved. Instead, the direct use of an as-


synthesized luminescent nanomaterial is preferred, particularly for POC assays, in order to reduce time, cost, and resources and to avoid extra steps. However, such approaches toward developing luminescence-based assays using nanoclusters on small molecules, produced through rapid synthetic routes to detect important cancer biomarker mucins, have not been pursued so far. In addition, the application of the same luminescent probe on small molecules for the specific labelling of cancer cells as well as tumor spheroids along with anticancer activity has not been explored.

In this chapter, a rapid synthetic route of luminescent phenylboronic acid templated gold nanoclusters (PB-Au NCs) to elucidate targeted anticancer therapeutic effects with simultaneous bioimaging and for application in in vitro POC diagnostics is reported. For theranostic applications, the PB-Au NCs probe was applied to specifically label (imaging) cancer cells by virtue of luminescence from Au NCs and simultaneously induce therapeutic effects toward cancer cells, resulting from the contribution of a boron component. The penetration and therapeutic efficacy of PB-Au NCs was also studied in the case of multicellular spheroids of HeLa cells to better mimic the tumor environment.

The uptake and detailed mechanism of cell death induced by PB-Au NCs were evaluated. Further, the antibacterial potential of PB-Au NCs was explored in order to assess its potential in combating secondary bacterial infections in cancer. Toward diagnostics, we discovered a rapid “one-step”

luminescent assay for mucin detection based on its interaction with an indigenously developed PB- Au NC probe and further developed a mobile-phone-based POC device with software for integration of the assay and readout. The specificity of the probe toward mucin resulted in the enhancement of luminescence upon its addition and thereby allowed one to develop a rapid assay to be integrated into a POC device. The overall work is illustrated in Figure 5.1 which depicts multiple applications of the as-synthesized PB-Au NC probe for mucin detection using a smartphone-based platform, targeted bioimaging, therapeutic activity toward cancer cells, and multicellular spheroids.

Figure 5.1. Schematic representation of the rapid synthesis procedure of PB-Au NCs and their application in targeted cancer

cell imaging and therapy as well as smartphone-based mucin detection.


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