*5.1. Biosensors*

A biosensor is an analytical device employed to detect chemical substances and has recognition elements (biological components), such as enzymes, antibodies, microorganisms, and DNA [132,133]. There is a higher demand for cheap, easy, fast, and sensitive analytical biosensors [134]. Synthesized organic–inorganic hybrid nanoflowers have recently been used as novel biosensors with different classes of enzymes and metal ions because of their high surface area and sensitivity [135].

Zhu et al. produced hNFs for the fast detection of aqueous phenol using laccase enzyme and copper phosphate integrated into a membrane. The results showed the ability for the rapid on-site detection of phenol in water [55]. Zhang et al. reported the production of an electrochemical biosensor using Mn ions/protein hNFs. These hNFs were able to detect ractopamine and showed a high activity and an excellent electrochemical performance [98]. Additionally, copper/HRP hNFs could be used as a sensor for dopamine detection [31]. Sun et al. [80] used dual enzyme (HRP + GOx) hNFs as colorimetric sensors for glucose detection.

## *5.2. Environmental Applications*

Environmental pollutants are a rising concern worldwide. Multiple methods have been used to eliminate these pollutants in the environment. One of these methods is bioremediation, which utilizes microorganisms and enzymes to reduce the concentrations of these harmful pollutants to an acceptable range [136–139]. Although enzymatic treatment has been shown to be effective, it faces some challenges, including a low activity, stability, and sustainability. Recently efforts have been made to immobilize enzymes on different

support materials that can eliminate some of the challenges of using free enzymes [140,141]. However, traditional immobilization techniques can lead to a reduced activity of the enzyme compared with the free enzyme. Thus, enzyme immobilization of hNFs has been shown to increase the activity, stability, and reusability of the enzyme [24,37]. Yilmaz et al. produced hNFs from BSA/copper ions for the selective separation of cadmium (Cd) and lead (Pb) in water, cigarette, and hair samples. The method reported a low detection limit for Cd and Pb compared with the other existing methods [131]. The Rong group synthesized hNFs using laccase/copper ions and loaded them onto a treated Cu foil surface. These hNFs showed a high decolorization efficiency and rate on Congo red (CR) dye compared with the free enzyme. In addition, the hNFs showed a good stability and reusability [32]. Fu et al. [34] produced enzyme hNFs that could degrade 100% of bisphenol A within 5 min.

#### *5.3. Industrial Biocatalytic Applications*

As a result of the grea<sup>t</sup> activity, stability, and reusability of enzymes immobilized on hNFs, there has been increasing interest in their use in industrial biocatalytic applications, making them a prominent research area [142]. Zhang et al. [77] used hNFs made from a dual enzyme system GOx + lipase/copper ions for the epoxidation of alkenes. The hNFs showed an excellent reusability, retaining 82% of their activity after ten reaction cycles. First, hydrogen peroxide is produced by GOx from glucose and is then directly used by lipase. As a consequence of lipase, carboxylic ester is converted into peracid. After that, peracid is used in the epoxidation of alkenes.

Hybrid nanoflowers are also used in the food manufacturing industry [62,95]. Bai et al. and Xu et al. were able to synthesize two expensive rare sugars: L-ribulose and D-tagatose. These sugars are monosaccharides, but are very difficult to find in nature. Multiple chemical reactions have been employed to manufacture L-ribulose and D-tagatose from disaccharides and polysaccharides. They used L-arabinose isomerase as the organic component in hNFs and manganese ions [95] and copper ions [62] to produce sugars. The conviction rates were 50% and 61.88% for the manganese hNFs and copper hNFs, respectively. In addition, hNFs can be used in the brewing industry. hNFs made from αacetolactate decarboxylase (ALDC)/calcium ions can be used to inhibit diacetyl formation in beer, which results in a buttery off-flavor [92].

#### **6. Cost Assessment and Industrial Integration Aspects**

As discussed above in the representative sections/subsections, the development of immobilized enzyme-based biocatalysts using different nanoscale materials, including hybrid nanoflowers (hNFs), has gained remarkable interest. The rationale behind this rising research trend in immobilized enzyme-based biocatalysts is the unique structural, physicochemical, and functional promises that enzyme-loaded hNFs offer with industrial integration potentialities. However, early-stage cost assessment and market analysis of enzyme-loaded biocatalytic nano-constructs, both pristine and hybrid, are equally essential to govern their industrial appropriateness [143,144]. The overall cost of the enzyme-based product and market analysis can be made via life-cycle assessments (LCAs) and/or technoeconomic scrutinizes as powerful tools. By taking the added value of these tools, an array of immobilized enzyme-based biocatalysts have been or are being assessed for commercialscale biocatalytic processes [145,146]. More specifically, from the cost and sustainability considerations, LCAs provide deep insight into the material, energy consumption, wasteful protection, and deprotection points in order to ensure the sustainability of the entire industrial process. The cost-effective ratio related to the energy consumption, chemical inputs, wasteful protection, and deprotection steps involved in the traditional industrial processes can be controlled effectively by implementing immobilized enzyme-based biocatalysts, e.g., enzyme-loaded hNFs, which are recoverable and reusable. For example, in several industrial processes, a massive amount of heat energy is required in the form of steam to preheat the feedstock for treatment purposes. In contrast, this can be significantly reduced

by alternating the process with immobilized enzyme-driven treatment [144]. According to the Global Industrial Enzymes Market Size and Regional Forecasts 2020–2027, in 2019, the global enzymes market was worth \$8636.8 Million USD, which is projected to reach up to \$14,507.6 Million USD with a compound annual growth rate of 6.5% over the duration of 2020–2027 [147]. Conclusively, the large-scale industrial integration and/or scale-up deployment of immobilized enzyme-based biocatalysts technology requires a proper understanding of both technological and economic aspects, as well as a good perception of the larger market forces.

#### **7. Conclusions and Future Perspectives**

Since the discovery of organic–inorganic hybrid nanoflowers in 2012, the topic has become a prominent research area and numerous types of biomolecules/inorganic hybrid nanoflowers have been explored. These hybrid nanoflowers have recently shown their ability to become stable, easy, fast, efficient, and recyclable for different biomolecules, specifically enzymes immobilizing host platforms. Additionally, hybrid nanoflower applications have been extended to biosensor designs, environmental treatment, bioassays, and different industrial biocatalysis. Studies have shown that hybrid nanoflowers lead to an enhancement in the immobilized enzyme's catalytic activity, resulting from the higher surface area of hNFs, a reduced mass transfer limitation, and favorable enzyme conformation in hNFs. Another potential advantage is that in some cases, enzymes immobilized on hybrid nanoflowers behaved better than other immobilization platforms, especially in terms of their reusability. For example, lipase enzyme immobilized on hybrid nanoflowers showed an excellent operation stability as they retained up to 94.5% of their activity, even after eight cycles of reaction [35]. Similar results were obtained by Li et al., where they could efficiently use the lipase/calcium nanoflowers up to six times [148]. On the other hand, when lipase was immobilized on the Fe3O4@MIL-100(Fe) composite it lost around 15% of its activity after only five cycles [149]. Similarly, when the alcalase enzyme was immobilized on hybrid nanoflowers [40] and glyoxyl supports [150], the nanoflowers could be recycled efficiently up to seven times, as opposed to the agarose beads (only five times). In addition, HRP immobilized on NH2-modified magnetic Fe3O4/SiO2 particles was only able to do four cycles before losing most of its activity [151], whereas HRP immobilized on hybrid nanoflowers only lost 25% of its activity after six reaction cycles [49].

Although there has been a rapid increase in the use of hybrid nanoflowers in recent years, there are still some key challenges that need to be addressed. First, the interaction between the organic component (enzymes) and the inorganic component (metal ions) needs to be examined more intensively, providing more information about the hybrid nanoflower design with a well-retained biological activity. This will help with controlling the morphology as well as adjusting the properties of the synthesized hybrid nanoflowers. In addition, this would facilitate the production of new kinds of hybrid nanoflowers for a particular application purpose. In addition, the nucleation step in the inorganic phase is still not described quantitatively in the presence of different amounts of enzymes. Second, hybrid nanoflowers with multiple enzymes or dual enzyme systems have not been thoroughly investigated. Third, more research needs to be done for examining the production of hNFs in organic media, as most of the synthesized hNFs are in aqueous media. This would be beneficial for industrial biocatalysis applications. Finally, the industrial application of hybrid nanoflowers can expand to include energy application like fuel cell fabrication and biodiesel. In summary, we believe that organic–inorganic hybrid nanoflower research will expand the application arena of hNFs and lead to smart solutions for modern problems.

**Author Contributions:** Conceptualization, S.S.A.; data curation, K.A.A.-M., A.A. and I.S.; writing— original draft preparation, K.A.A.-M. and A.A.; writing—review and editing, M.B. and H.M.N.I.; supervision, S.S.A.; project administration, S.S.A.; funding acquisition, S.S.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are thankful to their representative universities for supplying funds for this work. Partial funding for K.A.A. was allocated by the PhD fund (no. 31S389 to I.S.) from the College of Graduate Studies, UAE University. Generous support from Khalifa University to S.S.A. (CIRA-2020-046) is also graciously acknowledged. Consejo Nacional de Ciencia y Tecnología (CONACYT) is thankfully acknowledged for partially supporting this work under the Sistema Nacional de Investigadores (SNI) program awarded to Hafiz M. N. Iqbal (CVU: 735340).

**Conflicts of Interest:** The authors declare no conflict of interest.
