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Objectives
Biosensing is the ability to detect and measure the levels of different chemical and biological molecules in living systems. The main goal of biosensing in medical applications is to facilitate the prevention and monitoring of diseases. Examples of molecules that can be monitored through biosensing include glucose, calcium ions, and neurotransmitters. Biosensors are faster, less invasive ways of obtaining important biological information.
The brain is a critical organ in multicellular organisms. In humans, the brain consists of billions of neurons that create multiplex networks that influence learning, behavior, intelligence, and memory. Therefore, understanding brain physiology is important for the understanding, diagnosis, and treatment of neurological complications. Previous methods to study the brain included imaging techniques such as positron emission tomography, functional magnetic resonance imaging, and x-ray computed tomography, among others. These techniques are limited by the ability to provide constricted spatiotemporal data about the brain. Electrophysiology and optical neuroimaging are other techniques that have been used in brain sensing. However, electrophysiology is highly invasive because it requires direct physical contact with rain tissues. Conversely, the use of light to investigate neuronal signaling has several benefits, for example, adjustable wavelength, sensitive detection, low invasiveness, and high spatial resolution (Chen, Truong & Ai, 2017).
Optical neuroimaging is an indirect way of looking into neuronal signaling, which requires the use of sensors, probes, or indicators. Dyes that are sensitive to calcium or voltage changes and fluorescent indicators have been used to capture brain signals in vitro and in vivo. However, dyes can result in non-specific staining and cannot be localized spatially or temporally. Conversely, fluorescent dyes are unsuitable for direct in vivo uses on human subjects because they need genetic delivery. Nanozymes are versatile artificial catalysts that have wide biomedical applications such as biosensing and targeted therapy. Furthermore, it has been shown that nanozymes can be used as biosensors in the brain of mice (Chen et al. 2016). The discovery of nanozymes and their potential for biosensing applications is an interesting area of research that can be exploited to enhance the understanding of human brain physiology and associated conditions. Therefore, the purpose of this paper is to explore the possibility of using nanozymes in biosensing of the human brain in vivo.
Background
Enzymes are biological catalysts that have vast applications in various fields, including medicine, the manufacturing industry, pharmaceuticals, and the food industry. Enzymes are naturally produced by living organisms as part of native proteins. The advantages of natural enzymes include high specificity for reactions, can be reused over again, and work at low temperatures such as those present at physiological conditions. However, the main problem encountered when using natural enzymes is their high cost of production. Other shortcomings include extreme sensitivity to pH, ionic strength, and temperature, which denature them and alter their catalysis. Contamination of the reaction vessel with other substances in industrial applications can also affect their reactions.
These problems have led to studies to find other alternatives to natural enzymes, which led to the discovery of artificial enzymes. Artificial enzymes can be described as man-made, organic molecules that are designed to recreate the active site of natural enzymes, thus affecting catalysis (Esmieu, Raleiras & Berggren 2018). There are many types of artificial enzymes, including cationic polymeric products, nanoparticles, graphene, and its oxide, porphyrins, cyclodextrins, metal complexes, and dendrimers (Wei & Wang 2013). Some uses of artificial enzymes encompass healing and disinfecting wounds, killing bacteria, and getting rid of biofilms. Enzyme mimicry, which is the capacity to copy the structure and function of natural enzymes, has played a central role in the development of these synthetic enzymes.
Nanozymes are nanostructures with enzymatic activities. They have distinct features compared to artificial and natural enzymes, which have made them useful in biomedical applications such as bioimaging, bioanalysis, diagnostic medicine, targeted treatment, immunoassays, growth of stem cells, biosensing, and removal of pollutants (Wei & Wang 2013). A specific example of bioanalysis is the detection of glucose in serum using cerium oxide’s ability to mimic the action of the natural enzyme catalase. In the medical field, the constant use of antibiotics in the treatment of bacterial infections has helped to cure numerous diseases. However, the extensive use of antibiotics has led to resistance, which is a serious threat to global health. Subsequent studies have shown that some natural enzymes have the potential to confer protection against bacterial infections (Chen et al., 2018). Nanozymes are sensitive to chromatographic techniques due to their ability to react with chromogenic substrates, thereby leading to the formation of colored products. This property has enhanced its use in biosensing applications.
One advantage of nanozymes is diverse surface chemistry features that ultimately modulate their catalysis. For example, gold nanoparticles can undergo different surface alterations to mimic the activities of enzymes such as catalase, superoxide dismutase, peroxidase, and oxidase (Chen et al., 2018). The second benefit of nanozymes is the ability to withstand a wide range of pH, temperature, and salt concentrations, which makes for effective antibiotics under harsh conditions. Other advantages include low production costs and large-scale preparation. A recent technology is using light to regulate the activity of nanozymes with real-time precision (Wu et al. 2019). The main disadvantage of nanozymes is that they have lower specificity than natural enzymes, which restricts their in vivo applications (Cheng et al., 2017). The development of highly specific nanozymes will circumvent this shortcoming, enhance biosensing and pave the way for groundbreaking studies in living cells and organisms. Ultimately, there will be improved diagnoses and targeted treatments using non-invasive nanozyme technologies.
Hypothesis
Studies show that nanozymes are amenable to changes in specificity by altering their surface properties (Chen et al. 2018). This process enables the fabrication of nanozymes with different enzymatic activities for various purposes. Furthermore, nanozymes are stable over a wide range of conditions such as pH, temperature, and ionic strength. This stability implies that they can be subjected to diverse conditions during in vitro optimization processes to determine the most appropriate reaction conditions (Wei & Wang 2013). Furthermore, nanozymes are reported to have been used successfully in biosensing applications. A number of studies have also been done to elucidate the kinetics and reaction machinery of nanozymes. All these factors point toward the possibility of customized nanozymes for specific biosensing applications.
It is hypothesized that nanozymes can be used as effective biosensors of the human brain in vivo. If highly specific nanozymes that target important brain biomarkers such as glucose and calcium are developed, it will be possible to sense the levels of these molecules and study the structure and function of the brain. Furthermore, given that nanozymes can respond to different light wavelengths and elicit diverse enzymatic activities, it will be possible to use the developed nanozymes to study different physiological processes of the brain by simply altering the wavelength of light applied.
Methodology
The study would be conducted in three key stages of nanomaterial synthesis, characterization, and optimization of conditions. The materials, methods, chemicals, and techniques are specified for each stage of the process. The imidazole framework (ZIF-8) would be used. An aqueous solution of 0.5M zinc acetate (2 ml) would be poured into a similar volume of 2M 2-methylimidazole. The mixture would be stirred at room temperature overnight, leading to the formation of a white precipitate, which would be centrifuged and cleaned using anhydrous ethanol and deionized water. The product would then be vacuum-dried for 12 hours at 80oC. The nanozyme hemin@ZIF-8 would then be prepared by assimilating a hemin molecule into the ZIF-8 framework during the first chemical reaction. About 20 μL of 20 mM hemin dissolved in dimethylsulfoxide would be added to the reaction mixture. Other nanozymes would be prepared in the same way by adding 30 μL of 20 mg/mL GOx aqueous solution, 20 μL of 20 mM hemin and 30 μL of 20 mg/mL GOx, and 20 μL of 20 mM hemin and 30 μL of 2 mg/mL GOx-FITC for GOx@ZIF-8, GOx/hemin@ZIF-8, and GOx-FITC/hemin@ZIF-8, in that order (Cheng et al. 2016).
Characterization of the nanozymes would then be done by assessing specific enzyme activities. For example, a peroxidase chromogenic substrate (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) would be used to determine the enzymatic potential of hemin@ZIF-8 by adding 5 μL of the nanozyme to Tris-HCl buffer with hydrogen peroxide and ABTS at a pH of 7 and incubating at body temperature (37oC) for 20 minutes. The nanozyme would then be separated from the product via centrifugation, followed by measurement of the product through colorimetric analysis. Conversely, the activity of INAzyme would be determined by quantifying the oxidation of glucose and ABTS. The in vitro activity of the nanozyme would be ascertained by subjecting it to varying glucose concentrations in artificial cerebrospinal fluid (aCSF). A simulation of the in vivo nanozyme activity would be done by sampling microdialysate from the striatum of the living human brain and measuring its glucose concentration as described by Cheng et al. (2016).
The final step of optimization would be done in an online sensing platform based on INAzyme activity. In vivo microdialysis would be coupled with a microfluidic chip and a fluorescent detection system. The nanozymes would be restrained within the microfluidic chip in a microchannel. Brain microdialysates would be sampled continuously via a pump using aCSF as the perfusion solution at a rate of 1 μL/min. This step would enable the fluorescent detection of glucose by supplying a peroxidase substrate such as an Ampliflu Red solution at the same flow rate. A T-joint would be used to mix the peroxidase substrate and microdialysates online. The mixture would then be sent to the microchip containing immobilized INAzyme to react and generate fluorescent resorufin for subsequent detection.
The above method is valid and reliable because it has been tested successfully on mice’s brains. Most human studies in clinical research are usually based on the success achieved through mice studies. Consequently, it is expected that the outcomes will compare favorably when using human samples. The potential limitation of the method is that differences in the specific affinities of human and mouse substrates could alter the outcomes. It would not be possible to conduct reperfusion surgery and living brain ischemia studies on human subjects.
Reference List
Chen, Z, Truong, TM & Ai, HW 2017, ‘Illuminating brain activities with fluorescent protein-based biosensors’, Chemosensors, vol. 5, no. 4, pp. 1-28.
Chen, Z, Wang, Z, Ren, J & Qu, X 2018, ‘Enzyme mimicry for combating bacteria and biofilms’, Accounts of Chemical Research, vol. 51, no. 3, pp. 789-799.
Cheng, H, Zhang, L, He, J, Guo, W, Zhou, Z, Zhang, X, Nie, S & Wei, H 2016, ‘Integrated nanozymes with nanoscale proximity for in vivo neurochemical monitoring in living brains’, Analytical Chemistry, vol. 88, no. 10, pp. 5489-5497.
Esmieu, C, Raleiras, P & Berggren, G 2018, ‘From protein engineering to artificial enzymes–biological and biomimetic approaches towards sustainable hydrogen production’, Sustainable Energy & Fuels, vol. 2, no. 4, pp. 724-750.
Wei, H & Wang, E 2013, ‘Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzyme’, Chemical Society Reviews, vol. 42, no. 14, pp. 6060-6093.
Wu, J, Wang, X, Wang, Q, Lou, Z, Li, S, Zhu, Y, Qin, L & Wei, H 2019, ‘Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II)’, Chemical Society Reviews, vol. 48, no. 4, pp. 1004-1076.
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