# Application of Organic-Inorganic Hybrids in Chemical Analysis, Bio- and Environmental Monitoring
## 4 The Role of Nanoanalytics and Nanometrology in Biosensors/Nanobiosensors Development
For example, in biosensors fabrication it was proven that the original electrode exhibits numerous cavities (see Figure 8) which can be readily transferred to the active end-layer of the final nanohybrid. Another limitation of the AFM technique is that it is impossible to visualize the changes in surface chemistry of the nano-object. Thus, the heterogeneous blend of polystyrene and low-density polyethylene was characterized exclusively in terms of changes in their height profile, Figure 9. Instead, the changes in chemical profile, the monitoring of the degree of surface impurities, the quantification of the deposited rates, and the verification of possible degradation or re-arrangement of polymer over the surface of the inorganic template would be a desirable alternative. Figure 8: AFM three-dimensional profiles of the blank screen-printed carbon electrode (SPCE) (**a**), modified with graphene oxide (GO) and the cofactors (NAD+) (**b**), an additional layer of the enzymes L-lactate dehydrogenase (L-LDH, enzyme) and diaphragcase (DIA) (**c**) and a membrane of cellulose acetate (CA) (**d**). The scanned area of the AFM images was \(2\times 2\) μm\({}^{2}\). Reproduced with permission from [102], Copyright American Chemical Society, 2019. Figure 7: Case study of geometry and pitch measurement of periodic structures obtained by atomic force microscopy (AFM): _left_—Illustration of light and AFM interaction with a periodic surface structure; _right_—typical profile images of line scans obtained with AFM. Reproduced and adapted with the permission from ref. [101]. Notably, quantification of deposition rates for a thin layer of modifier (polymer or biomolecule) regardless of fabrication approaches is usually achieved by means of AT- and BT-cuts quartz crystal microbalances (QCMs), Figure 10[104, 105]. Resonance frequency shifts represent the weight of deposited nano-dimensional OIH structures with nanogram precision. The mass of the deposited nano-based OIH can be estimated using the Sauerbrey equation [106]. Remarkably, the rate of deposition is a very important criterion toward the control of the structure of the thin hybrids, their properties, and synthesis reproducibility [105]. In addition, during monitoring of the deposition process, the true values of the thin film thickness can be readily obtained. Moreover, QCM-based measurement systems are well suitable for the construction of biosensors recognizing affinity interactions [107, 108]. Figure 10: The scheme of the quartz crystal microbalances (QCM) sensor (top) and resonator model (bottom). NOTE: x\({}_{q}\)—is the quartz wafer thickness; x\({}_{f}\)—is the film thickness; p\({}_{q}\)v\({}_{q}\) and p\({}_{f}\)v\({}_{i}\) are the acoustic impedance for the unit cross section area of the crystal and of the film, respectively. Adapted with permission of ref. [109, 110] Copyright Elsevier, 1987. Figure 9: AFM images of a heterogeneous blend of polystyrene and low-density polyethylene (LDPE) in air using lateral resonance (LR) modes. Height images acquired with the LR mode (**A**); corresponding phase images (**B**); corresponding vertical deflection (quasi-static) images (**C**). Height profiles along the scan lines in image A (**D**). Reprinted and adapted with the permission from [103]. Copyright RSC Pub, 2009. Although QCMs were considered as very precise measurement nanoanalytical systems, they greatly suffer from non-linearity and hysteresis. In addition, the dependence of the resonance frequency on temperature makes QCM a technique that is strongly affected by irreproducible environmental factors. Summarizing, metrology at the nanoscale is addressed for (i) dimensional analysis of thin films, and nanostructured objects, i.e., nanoparticulate surfaces [111; 112] and (ii) characterization of the homogeneity/inhomogeneity profile of bio-, organic, and inorganic components over the surface of OIH.