Free-standing Cu-based NanoZymes for colorimetric sensing of glucose in human urine
Creators: name: Naveen Prasad, Sanjana
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310101
subject: Analytical biochemistry
classificationCode: 310607
subject: Nanobiotechnology
classificationCode: 340108
subject: Sensor technology (incl. chemical aspects)

description: Glucose is one of the most critical metabolites in our body, and the abnormality in its concentration range is associated with a variety of diseases and disorders. Therefore, accurate sensing of glucose in different body fluids is of high biomedical significance. A commonly known such disease is Diabetes mellitus, which is increasing globally with an alarming rate. An important aspect of diabetes management is to regularly monitor glucose levels. Although glucose detection in blood is rather easy by using low-cost commercial devices, renal glycosuria is another important condition that is commonly observed in patients with extended period of high glucose levels or in Type I juvenile diabetes. This condition leads to the excretion of glucose in urine. This is also a common occurrence in patients with Fanconi syndrome, and many other disorders. As such, the urine glucose levels can be considered as reliable indicators for screening patients with high glucose levels. Urine glucose test strips are commercially-available, however, they suffer from limited sensitivity in the human body-relevant glucose concentration range. Further, these urine test strips are time-sensitive i.e. the color response varies even if the strip is read within 1 min error interval of the recommended time. This tends to lead to false-positives.<br><br>As such, the glucose monitoring tools typically employ a combination of glucose oxidase (GOx) and Horseradish peroxidase (HRP) enzymes for glucose detection. In this reaction, GOx oxidises glucose to produce gluconic acid and H2O2. The H2O2 is then either detected electrochemically (in commercial blood glucose monitoring strips) or it serves as a substrate for HRP to catalyse the conversion of a non-colored substrate to a colored product (in pathological tests). A potential drawback of this system is that the HRP can be easily inactivated by H2O2. A viable alternative to using HRP is more robust artificial enzymes. A recent discovery that certain nanoparticles can show enzyme-mimic activity (commonly referred to as NanoZymes) can offer a potential solution, wherein HRP is replaced with nanoparticles with peroxidase-mimic activity. While such solution-based NanoZymes have shown promise in glucose sensing, they are limited to detecting pM to µM concentrations of the analyte, while the concentration of glucose in urine is in the mM range. Keeping this aspect in mind, this thesis attempts to develop a sensing system that can detect glucose in the biologically-relevant range. This is achieved by loading catalytically active copper nanoparticles as NanoZymes on high surface area templates such as cotton fabric (Chapter 3), and subsequently further improving the ability to detect glucose colorimetrically by creating free-standing bimetallic NanoZymes on the surface of cotton fabric (Chapter 4).<br><br>In the first working chapter of this thesis (Chapter 3), the outstanding catalytic properties of copper nanoparticles embedded within the 3D matrix of cotton fabric (Cu@Fabric) is established. This is the first time that the catalytic activity of a NanoZyme is observed to result in the generation of the second oxidation product of the peroxidase substrate, TMB (3,3',5,5' tetramethylbenzidine) at mildly acidic conditions. Notably, this process typically requires highly acidic conditions (pH 1). The absorbent and porous nature of the template in combination with the inherent high catalytic activity of copper nanoparticles appears to be responsible for this outstanding catalytic performance. Considering the high catalytic activity, the HRP in the typical glucose sensing system is subsequently replaced with the Cu@Fabric NanoZyme to effectively quantify glucose in the biologically-relevant concentrations even in the presence of complex biological matrix of urine.<br><br>To further improve the colorimetric response and stability of the Copper@Fabric (decrease the leaching of the copper during assay), in Chapter 4 of this thesis, the copper is galvanically replaced with small quantities of noble metals to create bimetallic fabrics. Considering that bimetallic nanomaterials display enhanced catalytic properties over their individual counterparts, the bimetallic fabrics obtained after the galvanic replacement reactions showed improved peroxidase-mimicking catalytic activity. Among the four bimetallic systems (Cu-Au, Cu-Ag, Cu-Pd and Cu-Pt), the Cu-Pt@Fabric NanoZyme showed the highest initial rate of the reaction. The formation of the bimetal also reduced the surface oxidation of copper as well as the leaching of Cu ions during glucose sensing assays. The improved stability resulted in higher recovery and reduced standard deviation of the glucose sensing system in comparison to the pristine Cu@Fabric used in Chapter 3. The bimetal system also showed a more intense colorimetric response which is attributed to the fact that the bimetallic NanoZyme system did not favour the double oxidation of TMB.<br><br>Overall, this thesis makes an important contribution towards highly accurate, user-friendly colorimetric sensing of glucose in urine in biologically-relevant range, which is likely to be of high clinical and commercial interest.