Towards the development of surface-confined fluorescence boronic acid-based sensors for saccharide detection

Buccoli, Laura (2023). Towards the development of surface-confined fluorescence boronic acid-based sensors for saccharide detection. University of Birmingham. Ph.D.

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Abstract

Carbohydrates (saccharides) are among the most abundant organic compounds in nature. Due to their diversity and the ability to conjugate with themselves or other molecules, they create an enormous variety of compounds called glycans in biological settings. Glycans are involved in several physiological events inside the body. The discovery of abnormal concentrations of glycans can be indicative of disease, making their detection useful for early diagnosis and treatment. Therefore, research on glycan sensing systems has become substantial in recent years. One of the more interesting strategies is the use of boronic acids, which have the ability to form complexes with diols. Several boronic acid sensing systems have been designed to improve the sensing efficiency. These include strategies such as supramolecular aggregation, multicomponent covalent assembly, and the use of new materials such as polymer gels, nanoparticles, or quantum dots. Due to the quick response time, low cost, and local observation capabilities, fluorescence techniques have been widely employed in sensing applications. Boronic acids (BAs) are used in conjunction with dyes to modulate the signal upon saccharide binding through fluorescent mechanism as Photoinduced electron transfer (PET), Internal charge transfer (ICT), Forster resonance energy transfer (FRET), or their combination. The performance of the sensor has been demonstrated to be enhanced by its attachment to a solid surface. The effect of the surface is to reduce the matrix interference and bring close the system units strengthening their interaction and so the fluorescence mechanism involved. Moreover, the sensitivity could be improved by incorporating multiple sensors on the surface. Although different materials such as quantum dots, polymers, and hybrid nanogels have been functionalized with BAs, boronic acid-based sensors designed to be surface- confined have not been extensively investigated in research associated with saccharides detection.

The aim of this work was to design fluorescence boronic acid sensing systems to detect sugars that could be immobilized onto solid supports for high-sensitivity saccharide detection. Different boronic acid-based sensing systems with fluorescent capabilities were investigated. In conjunction, the functionalization of silicon wafers and silica nanoparticles was explored as solid supports for the immobilization of the fluorescence systems.

The first sensing system consisted of two single units: phenylboronic acid as a receptor and an anthracene dye as a fluorophore. The anthracene fluorescence was expected to be modulated by a PET mechanism due to the quenching effect associated with the boronic acid. Saccharide binding will then suppress this mechanism, amplifying the response. The interaction between units can be strengthened by immobilising the sensor system onto a silica surface, thereby promoting the PET mechanism, and further improving the signal response. However, in contrast to that found in the literature, a fluorescence quenching due to the presence of the boronic acid was not observed. It was therefore concluded that PET was not involved in the first designed sensor system.

To promote the interaction between the receptor and fluorescent units, the second sensing system involved a pyrene-based sensor with BA unit incorporated into the structure of the fluorophore. To evaluate the affinity towards sugars and the fluorescence response two strategies were investigated: direct connection into the structure of the fluorophore or through an alkyl chain with an amino group as a linker. Moreover, since FRET mechanism can occur at longer distances (10 to 100 Å) compared to PET, it was considered an alternative fluorescent mechanism. Coumarin was selected as a probe to act as acceptor in FRET couple with the second sensing system acting as donor. The change in fluorescence intensity caused by saccharide binding would lead to the interaction between 1-boronic acid pyrene and coumarin, promoting FRET. However, addition of sugar resulted in a minor fluorescence intensity increase, insufficient to cause an efficient

interaction between the FRET couple. To produce the sufficient change in fluorescence required to induce FRET, a pyrene-based sensor, involving an alkyl chain with an amino group as a linker with phenylboronic acid receptor, was synthesised using another approach. The fluorescence of this designed sensor was planned to be modulated by ICT mechanism. A noticeable shift in the emission spectra was expected upon sugar binding, enabling FRET via coumarin 9. Despite multiple attempts, the presence of side reactions and issues with purification meant the designed sensor could not be synthesised successfully. Experiments were therefore conducted with a derivative, which displayed a higher fluorescence intensity increase compared to the previous system. However, evidence in support of an ICT mechanism occurring was not found. It was concluded that the structure of the sensing system required improvement to promote the fluorescence mechanism involved. Focus was also given to a different mechanism which is less affected by matrix composition, in comparison to ICT, hoping to achieve the required fluorescence intensity.

The third system aimed to strengthen the weak interaction between units seen in the previous systems, which resulted in the unsatisfactory fluorescence signals seen. It focused on the structure of the sensor towards the presence of a right linker and a quenching group in a strategic position, aiming to achieve a higher sensitivity for saccharide binding. The third system involved a pyrene- based sensor with the boronic acid connected through a piperazine ring. Piperazine acts as both as a linker and a fluorescence quencher due to its amino group, ensuring a PET process is involved. The sensor was also functionalized with an acetylene group to allow for immobilization onto surfaces. The synthesis of final sensors was performed employing a definitive combination and sequence of reactions. Performed studies with the final system showed a quenching behaviour of the fluorophore, which was thought to be due to the length and rigidity of linker, absent in the

previous system. The synthesized compound displayed fluorescence enhancement after sugar addition in the millimolar range. This confirmed that the assumed PET mechanism was involved, and further suppressed to restore the fluorescence abilities of pyrene. The fluorescence intensity change produced by the saccharides binding by PET was then successfully employed to establish the interaction with an alternative probe, attesting to a FRET process.

The systems are planned to be immobilized onto silica surfaces such as silicon wafers or silica nanoparticles. The functionalization of silicon wafers with different silanes was achieved successfully by self-assembled monolayer (SAM) formation. However, there was a considerable degree of inter-chip variation and poor reproducibility. Silica nanoparticles (SiO2 NPs) were selected as an alternative solid support due to their reported use in sensing applications. The azide group was incorporated in amino-functionalised SiO2 NPs for future sensor inclusion.

The boronic acid drives the action in this sensing system and the presence of an amino group improves the affinity of boronic acid towards saccharides. Moreover, this interaction was strengthened by employing a linker with a semi-rigid structure such as piperazine. Having an amino group in a strategic position acting as a quencher, strengthened the PET mechanism towards the fluorophore. The fluorescence response achieved following saccharide binding could be employed to establish the interaction with an additional probe and enable a FRET mechanism. Since the versatility in sensing application and the wide range of techniques available for their characterization, SiO2 NPs have been selected as solid support for the incorporation of sensing systems. The azide functionalized surface will enable the immobilisation of the system through click chemistry by acetylene-azide reaction.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):