Nanobiosensors based on hybrid materials

Nanobiosensors based on nanohybrids and nanocomposites have attracted increased attention because they provide enhanced sensitivity, selectivity, robustness, and simplicity to the functional interfaces (^{Cajigas & Orozco, 2020}; ^{Soto & Orozco, 2022b}). Nano-composites combine two or more materials of different properties resulting in novel physicochemical properties with one of the dimension constituents at the nanoscale or, instead, the nanocomposite structure exhibiting a nanometric phase separation of the individual components. Therefore, nanocomposites present mixed properties based on the original properties of each nanomaterial constituent (^{Ashby et al., 2013}). Similarly, organic and inorganic building blocks are combined in hybrid nanomaterials (^{Alemán et al., 2007}) with a serial interface between the structural components (^{Ashby et al., 2013}) and emerging improved physicochemical properties distinct from the components alone. Metallic nanostructures (^{Arduini et al., 2016}), silicon nanomaterials (^{Ahmed et al., 2021}; ^{Hasanzadeh et al., 2012}; ^{You et al., 2020}; ^{Zhang et al., 2013}), carbon nanostructures, and semiconductor polymers (^{Barsan et al., 2015}; ^{Duan et al., 2016}; Y.^{Liu et al., 2015}; ^{Noreña-Caro & Álvarez-Láinez, 2016}; ^{Promphet et al., 2015}) are the most common hybrid nanomaterials applicable to electrochemical biosensing to date.

Recently, we reviewed the role of hybrid materials on nanobiosensors (^{Cajigas & Orozco, 2020}; ^{Soto & Orozco, 2022b}). Besides, a multifunctional hybrid material was developed based on iron NPs coated with graphene and supported on carbon nanotubes. The new hybrid was exhaustively characterized from the material (^{Gallego et al., 2017}) and electrochemical (^{Soto et al., 2018}) points of view. For this purpose, the material was deposited on the surface of a screen-printed carbon electrode (SPCE) (Figure 2A) (^{Soto et al., 2018}), and its electrochemical properties were evaluated in each assembly stage. The results showed that the hybrid material had improved electrochemical properties compared to each component acting alone. In addition, the outer layer of graphene, covering the iron NPs, served as protection to prevent oxidation and maintain its catalytic properties for longer. Finally, it was clear that the electrochemical response of the hybrid material was proportional to changes in the concentration of hydrogen peroxide (H₂O₂) in the order of mM and had a higher sensitivity compared to the response of the individual components.

Figure 2 Nanobiosensors based on hybrid materials. A) Multifunctional hybrid material based on iron NPs coated with graphene and supported on carbon nanotubes for H₂O₂ sensing (Reproduced with permission, Copyright © 1999-2023 John Wiley & Sons, Inc. B) Catalase (Cat)-functionalized hybrid nanomaterial to detect H₂O₂ with enhanced sensitivity (Reproduced with permission, Copyright © 2023, Elsevier B.V.) 

The conjugation of biomolecules with hybrid nanomaterials has offered opportunities to assemble nanobiosensors of much more improved electrochemical performance. In this context, to improve the analytical performance of the first nanohybrid structure, a novel catalase (Cat)-functionalized hybrid nanomaterial was developed to detect H₂O₂ with enhanced sensitivity (Figure 2B) (^{Soto et al., 2021}). The rationally assembled nanobiosensor used Fe@G-MWCNTs deposited at an SPCE functionalized with Cat by covalent coupling. TEM, X-ray diffraction-, X-ray photoelectron- and thermogravimetric analysis were used to characterize the physicochemical properties of the hybrid nano-biomaterial and better understand its electrochemical behavior. The results evidenced enhanced detection of H₂O₂ (linear range from 0.1 to 7 mM, limit of detection (LOD) of 28.2 pM and sensitivity of 0.059 pA/(pM.cm²) concerning the hybrid nanomaterial laking Cat (linear range of 0.5 to 9.8 mM, LOD 0.65 mM and sensitivity of 7.97 pA/(mM.cm²).

Nanobiosensors for the detection of pathogens

Along with multiple-target analytes and biomarker monitoring, pathogen detection is paramount in precision medicine. It is part of the arsenal of tools required to know the status of a patient affected by a disease. Furthermore, pathogen detection searches to identify specific microorganism biomolecules or molecular changes in the host (^{Zhang & Guo, 2020}). In the following paragraphs, some cases illustrating the role of pathogen detection in precision medicine it is presented. Electrochemical nanobiosensors have been used to detect viruses (^{Alzate et al., 2020}; ^{Alzate, et al., 2022}; ^{Alzate, et al., 2022}; ^{Cajigas et al., 2020}, 2022), parasites (^{Echeverri et al., 2020}) and bacteria (^{Vásquez et al., 2017}) with high sensitivity, low LOD, and straightforwardness, demonstrating their potential for disease diagnosis and monitoring the host-pathogen ecosystem.

For example, we developed a dual electrochemical magneto-nanogenosensor for differential diagnosis of the Zika virus and its discrimination from homologous arboviruses such as dengue and chikungunya (^{Alzate et al., 2020}; ^{Alzate et al., 2022}; ^{Cajigas et al., 2020}). The diagnostic tool integrated magnetic nanoparticles as a support platform with highly selective DNA strands as bioreceptors, which responded to changes in the concentration of Zika genetic material. The nanogenosensor was assembled in a sandwich format, as shown in figure 3A. A DNA strand anchored on the surface of the magnetic nanoparticles A(1) hybridizes the genetic material of the target virus with a DNA strand (2) that is labeled with an enzyme attached to an antibody (3). The resulting nanobioconjugate was transferred to a screen-printed gold electrode (SPAuE) (B) for the corresponding electrochemical reading (C). The enzyme produced an electrical current proportional to the concentration of the virus's genetic material present in samples from the patients. It was possible to sensitize the magnetic nanoparticles with specific DNA probes for Zika, Dengue, or Chikungunya, which, once transferred to the SPE compartments, allow the discrimination of the Zika virus from its homologs in a single test.

Figure 3 Nanobiosensors for the detection of pathogens. A) Dual chronoamperometric magneto-nanogenosensor for differential diagnosis of the Zika virus and its discrimination from homologous arboviruses such as dengue and chikungunya. B) Electrochemical immunosensor for amperometric detection of SARSCoV-2. C) Electrochemical biosensor based on peptides immobilized on SPAuEs for the rapid and specific detection of unlabelled spike protein from SARS-CoV-2 by EIS. D) Electrochemical genosensor for detecting viral RNA from SARS-CoV-2 by chronoamperometry. E) Highly sensitive and specific electrochemical genosensor to detect genotype 3 of hepatitis E virus (HEV) in wastewater samples by chronoamperometry. F) Synthetic glycosylphosphatidylinositol-based biosensor for the detection of toxoplasmosis by EIS. G) First amperometric immunosensor for the rapid detection of Streptococcus agalactiae (A: Sketch of the concept with all the assembling steps from 1 to 5. B: Typical chronoamperometric layout, and C: SEM image of bacteria confined at an SPCE surface. (All images were reproduced with permission, Copyright © 2023, Elsevier B.V.) 

An electrochemical immunosensor was recently developed for the amperometric detec tion of SARS-CoV-2 (V.^{Vásquez et al., 2022}). The sandwich-type immunosensor used magnetic particles that take advantage of the high-affinity spike-ACE2 proteins interaction and a poly-horseradish peroxidase (HRP) enzyme complex to amplify the resultant signal. The particles were confined at SPAuE and the reaction was followed by chronoampero-metry reaching 22.5 ng/mL LOD in 5 uL samples. The device's high performance was also tested in a pocket potentiostat (Figure 3B) with identical results compared to a laboratory potentiostat in line with the concept of POC. For this same purpose, the first electrochemical biosensor based on peptides immobilized on SPAuEs was developed for the rapid and specific detection of unlabelled spike protein from SARS-CoV-2 by electrochemical impedance spectroscopy (EIS) (^{Soto & Orozco, 2022a}). The biosensor obtained an 18.2 ng/mL LOD in spike protein standard solutions and 0.01 copies/mL of lysed particles in 15 min (Figure 3C), which is considered clinically relevant. It is noteworthy that both devices detected the SARS-CoV-2 in positive samples (by RT-PCR) with no signal in negative samples (from healthy individuals) highlighting the potential of the devices to detect the spike protein from SARS-CoV-2 and viral particles from clinical samples.

Using magnetic particles as a supporting platform, an electrochemical genosensor was developed by covering it with thiolated capture probes (Figure 3D) for detecting viral RNA from SARS-CoV-2. The RNA was sandwiched between the capture and biotinylated signal probes modified with enzyme complexes achieving an 807 fM LOD and high specificity to discriminate from SARS-CoV, MERS, and HKU1 homologous viruses (^{Cajigas et al., 2022}). These examples demonstrate the significant advantages of applying electrochemical biosensors to different molecular levels in SARS-CoV-2 infection as a diagnostic component in COVID-19 precision medicine.

The following example describes a highly sensitive and specific electrochemical genosensor to detect genotype 3 of the hepatitis E virus (HEV) in wastewater samples (Figure 3E) (^{Alzate et al., 2022}). Highly specific DNA target probes were designed to hybridize a target sequence of HEV between a biotinylated capture probe and a labeled signal probe in a sandwich-type format. An enzyme-labeled antibody allowed straightforward electrochemical detection. The specificity of the probes was determined in silico and by PCR and qPCR assays demonstrating efficient amplification of two targets. The genosensor electrochemical response was target concentration-dependent from 300 pM to 2.4 nM with a sensitivity of 16.93 LiA/nM, a 1.2 pM LOD, high reproducibility, and differential against HEV genotype 3 viral genomes.

For the detection of toxoplasmosis, a transducer platform was functionalized with a synthetic glycosylphosphatidylinositol (GPI) glycan (Figure 3F) (^{Echeverri et al., 2020}) distinguishing disease states in human sera (^{Götze et al., 2014}). This means that a label-free electrochemical glycobiosensor for detecting anti-GPI IgG and IgM antibodies in serum from toxoplasmosis seropositive patients was established. This biosensor used the synthetic GPI phosphoglycan bioreceptor anchored at the SPAuE through a linear alkane thiol phosphodiester. The antigen-antibody interaction was detected and quantified by EIS. The resulting device showed a linear dynamic range of anti-GPI antibodies in serum ranging from 1.0 to 10.0 IU mL^-1, with a 0.31 IU mL^-1 LOD. The method also has great potential for detecting IgG antibodies related to other multiple medical conditions characterized by antibody overexpression. Moreover, the electrochemical biosensor has the additional advantage of a simple, portable, and cheaper tool for a closer-to-the-patient diagnosis.

The first amperometric immunosensor for rapidly detecting Streptococcus agalactiae (^{Vásquez et al., 2017}) is a helpful example of detecting a pathogen in its ecosystem. The biosensor consists of a biotinylated antibody (AT) (Figure 3G) with two functions: to selectively interact with the antigens expressed in the bacterial cell wall (A2) and to immobilize the resulting bioconjugate (A3) at the SPEs surface coated with neutravidin (A4). When the bioconjugate reacts with the streptavidin-HRP complex (A5), an electrochemical signal was produced proportional to the concentration of bacteria present in a given sample (Figure 3G, B, signal in blue) and to the minimum signal generated with a control sample (signal in black). Figure 3G, C shows an electron microscopy (SEM) image with the bacteria confined at the SPE surface. The immunosensor allowed the selective detection of S. agalactiae cells compared with the response in samples containing other bacteria that can coexist in the same environment in 90 min. Highly sensitive quantification of bacteria was also achieved in a concentration range of 10¹ to 10⁷ CFUml^-1 with a 10 CFUml^-1 LOD in buffered solutions doped with bacteria and the detection of bacteria in water samples from a lake in Huila, Colombia (2°41'6"N, 75°26'24"W), demonstrating its practical utility.

These examples show the detection of different pathogens for disease diagnosis and in environmental matrixes potentially useful for surveillance systems and disease control in a specific population.

Nanobiosensors for the detection of cancer-related biomarkers

Smart biosensors may play a crucial role in developing decentralized analysis systems that can bring pathogen detection and microorganism diagnostic capabilities from laboratories to remote settings and even at home. They also have promising testing solutions to monitor other disease-related biomarkers. For example, nanobiosensors for detecting a panel of biomarkers of colorectal cancer (CRC) offer opportunities for early diagnosis, prognosis, predictions of the course of the disease, metastasis, or response to treatment (^{Quinchía et al., 2020}). For this purpose, selecting the panel of biomarkers is crucial, i.e., ideally, biomarkers of different molecular levels in line with the concept of precision medicine.

In the context of nanobiosensors for CRC-related biomarker detection, we reported the first electrochemical immunosensor for detecting p-1,4-GalT-V (Figure 4A) (^{Echeverri & Orozco, 2022a}). A label-free electrochemical immunosensor coupled an anti-p-1,4-GalT-V antibody covalently at a mixed self-assembled monolayer (SAM)-coated SPAuE surface. This sensitized platform captured the p-1,4-GalT-V biomarker from human serum samples with high specificity. The clinically relevant response, monitored by EIS, was protein concentration-dependent showing a linear dynamic range from 5 to 150 pM, with a sensitivity of 14 Q pM^-1 and a 7 pM LOD. This outstanding performance highlights its great potential for including it in a biomarker signature for the early diagnosis/prognosis of CRC.

Figure 4 Nanobiosensors for the detection of cancer-related biomarkers. Electrochemical immunosensor for detecting P-1,4-GalT-V by A) EIS and B) ECS. (Reproduced with permission, Copyright © 2023, Elsevier B. C) Nanocomposite-decorated SPAuE for the label-free electrochemical detection of anti-p53 autoantibodies by DPV. D) Poly(thiophene acetic acid)/Au/poly(methylene blue) nanostructured interface at SPCE for label-free detection of p53 protein by SWCV. (Reproduced with permission, Copyright © 2023 Springer Nature Switzerland AG.) 

However, to increase the sensitivity and reduce the LOD of this biosensor, we reported the first capacitive nanobiosensor for detecting p-1,4-GalT-V (Figure 4B) (^{Echeverri et al., 2023}). To this end, we nanostructured the surface of the SPCEs with gold nanorods (AuNRs) and Prussian blue (PrB). AuNRs increased the SPCE surface area for physical adsorption of the capture antibody and promoted the deposition of PrB as a redox-active compound at the SPCE surface. The molecular biorecognition event perturbed the PrB redox density-of-states changing surface capacitance (Cu) that correlated well with increasing biomarker concentrations from 50 to 400 fM, 1.5 uF fM^-1 cm^-2 sensitivity, and 20 fM LOD. The label-free reagentless electrochemical device detected the glycoprotein by electrochemical capacitance spectroscopy (ECS) in unpretreated human serum samples with high specificity and ultrasensitivity. The device can be used as a CRC diagnosis/ prognosis tool in a decentralized setting with minimal patient sample manipulation and rapid response.

It is clear that detecting autoantibodies (Aabs) can be a potential approach with predictive value for early cancer diagnosis and preventive and curative treatments before the tumor progresses to the late stages (^{Tan et al., 2009}). Thereon, we reported a nanocomposite-decorated SPAuE for the label-free electrochemical detection of anti-p53 autoantibodies (Figure 4C) (^{Cruz-Pacheco et al., 2022}). The nanoimmunosensor was prepared by in situ electropolymerization of 3,4-ethylenedioxythiophene (EDOT) on SPAuE in the presence of CeO₂ NPs. The p53 antigen was covalently linked to the resul tant Ce/PEDOT/SPAuE platform for determining anti-p53 autoantibodies by differential pulse voltammetry (DPV) in a linear range from 10 to 1000 pg mL^-1 and with a 3.2 pg mL^-1 LOD. The nanoimmunosensor offered high specificity, selectivity, and long-term storage stability with great potential to detect anti-p53 autoantibodies in serum samples. Incorporating organo-functional nanoparticles into polymeric matrices can provide a simple-to-assemble, rapid, and ultrasensitive approach for on-site screening of anti-p53 autoantibodies and other disease-related biomarkers with low sample volumes.

Increased serum levels of the tumor suppressor protein p53 have become a promising biomarker for diagnosing CRC (^{Aydin et al., 2018}). We electrochemically assembled step-by-step a poly(thiophene acetic acid)/Au/poly(methylene blue) nanostructured interface at SPCE for label-free detection of p53 protein (Figure 4D) (^{Cruz-Pacheco et al., 2023}). The conductive properties of the polymeric interface increased with an additional layer of poly(methylene blue) electropolymerized in the presence of gold nanoparticles. Anti-p53 antibodies were covalently immobilized through the poly(thiophene acetic acid) moieties at the resultant nanoarchitecture as bioreceptors. Under optimal conditions, p53 was specifically and selectively detected by square-wave cyclic voltammetry (SWCV) in a linear range between 1 and 100 ng mL^-1 with a LOD of 0.65 ng mL^-1. In addition, the electrochemical nanoimmunosensor detected p53 in spiked human serum samples and CRC cell lysates; the results did not exhibit significant differences compared to standard spectrophotometric methods. The resultant p53 nanoimmunosensor is simple-to-assemble, robust, and has the potential for point-of-care biomarker detection applications.

Nanobiosensors for diagnostics, prognostics, and assessing the risk of complications in patients

Some biomolecules can indicate the presence, severity, or type of disease. These biomolecules are commonly named biomarkers and play a fundamental role in diag nosing and predicting disease severity and future complications (^{Zhang & Guo, 2020}). However, unlike single biomarkers, a panel of biomarkers should be analyzed in the con text of precision medicine as commented. For example, pathogen detection requires theidentification of specific microorganism biomolecules or host molecular changes, including detecting RNA, antigens, host-generated antibodies, or whole cells. This information can be combined with monitoring inflammatory, hematological, biochemical, and endothelial biomarkers, among others, searching to elucidate different pathways of clinical manifes tation of a disease or disease complications such as a high inflammatory response, low white blood cell and lymphocyte counts, and abnormal coagulation parameters. Such complications are associated with proteins and genetic factors expressed differently in each person and with disease progression and severity (V.^{Vásquez et al., 2022}).

In this sense, it is worth mentioning the cytokine storm inflammatory response produced in response to, for example, viral infections and their progression (^{Alosaimi et al., 2020}). Excessive release of inflammatory biomarkers in response to infections may trigger sepsis and complications, including lung injury and, eventually, fatal outcomes (^{Kumar et al., 2020}). Furthermore, changes in inflammatory cytokines levels such as interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), and tumor necrosis factor-alpha (TNF-a) and chemokines such as C-X-C motif chemokine ligand 10 (CXCL10), chemokine ligand 3 (CCL3), and monocyte chemoattractant protein 1 (MCP1) have been related with severe infections (^{Liu et al., 2020}; ^{Mariappan et al., 2021}). High levels of IL-6, IL-10, and TNF-a have also been reported in patients with post-acute sequelae of infection after diagnosis (^{Peluso et al., 2021}). Finally, while vitamin D is a biomarker most frequently related to susceptibility to infections, lymphocyte count (lymphopenia), procalcitonin (PCT), IL6, and C-reactive protein (CrP) have been related to disease severity, and the D-dimer, cTn, and LDH biomarkers to the risk of death, (^{Malik et al., 2021}).

Although they are not positioned at the clinical level yet, nanobiosensors have shown outstanding potential not only for diagnostics but for prognosis and determining the course of the disease at the research level. For example, a low-cost, portable, and wireless multiplexed biosensor platform was reported for the rapid and ultrasensitive detection of nucleocapsid proteins as indicative of viral infection and IgG and IgM antibodies of the immune response and CrP of disease severity (^{Torrente-Rodríguez et al., 2020}). In relevant physiological ranges, the device also showed a highly selective and rapid response (1 to 10 min) in blood and saliva samples. This illustrates the implementation of electrochemical biosensors at different molecular levels for diagnosis, progression, and disease severity in line with precision medicine.

Nanocarrier-based therapeutics

Proper detection and timely diagnosis are at the forefront of measures to face diseases. However, success depends on other essential processes that include the selection of proper drug regimens involving the type of drug, dose, and frequency (^{Sánchez et al., 2020}). Encapsulating drugs into biocompatible and biodegradable polymeric nanocarriers, among nanocarriers from other materials, helps to overcome the limitations related to high lipophilicity, low water solubility, and poor biodistribution of some drugs. Furthermore, the loading capacity and encapsulation efficiency can be modulated depending on the formulation's polymer characteristics, synthesis method, and other components. Besides, this strategy protects the cargo from degradation and reaction with other drugs or molecules in the human body in its journey to the therapeutic target. Therefore, it is expected to improve the efficiency and efficacy, decrease the doses and frequencies required to reach the therapeutic doses, and increase its effectiveness, thereby preventing the appearance of side effects and the development of, for example, the resistance of microorganisms to therapeutic agents.

Most often, nanosystems utilized in drug encapsulation include lipid nanoparticles, nanoemulsions, nanosuspensions, nanogels, liposomes, niosomes, and polymeric nano-particles (^{Colorado et al., 2020}). Among these, niosomes are vesicular systems that have drawn attention due to their chemical stability, cost-effectiveness, and preparation simplicity without harsh solvents (^{Aditya et al., 2017}; ^{Fidan-Yardimci et al., 2019}). In this sense, it is worth mentioning our evaluation of the metabolic activity of anthocyanins (ACNs) encapsulated into niosomes in a diet-induced murine obesity model to increase their oral bioavailability. The ACN-loaded NPs with low dispersity, a negative surface charge, and 57 % encapsulation efficiency ameliorated insulin resistance and glucose intolerance and reduced animal weight and plasma insulin, glucose, leptin, and total cholesterol levels in obese mice.

Often, polymeric nanocarriers are also a choice when encapsulating drugs. Properties of nanocarriers from synthetic polymers can be readily tunned on demand by modulating molecular weight, polymer concentration, and polymer composition using surfactants and/ or energy inputs (or not), depending on the fabrication method. In this field, aiming to contribute to a new arsenal of alternatives to fight intracellular infections, we developed a therapeutic strategy based on encapsulating itraconazole (ITZ) into functionalized NPs for their targeted and controlled release into macrophages (Figure 5A) (^{Mejía et al., 2021}). NPs were based on poly (lactic acid-co-glycolic acid) (PLGA) polymers of different compositions, molecular weights, and lactic acid-to-glycolic acid ratios self-assembled by the high-energy nanoemulsion method. Results showed improved drug-loading capacity and encapsulation efficiency by lowering the pH and using a mixture of surfactants, initial immediate ITZ release, followed by a prolonged release phase that fitted better with a Fickian diffusion kinetic model and high thermal stability. In addition, NPs were also stable, efficient, and reproducibly functionalized with F4/80 and mannose by the carbodiimide approach (Mejía et al., 2022). Overall, in-vitro assays showed the nanosystem's efficacy in eliminating the Histoplasma capsulatum fungus and paved the way to design highly efficient nanocarriers for drug delivery against intracellular infections.

Figure 5 Nanocarrier-based therapeutic. A) Therapeutic strategy based on encapsulating itraconazole (ITZ) into functionalized NPs for their targeted and controlled release into macrophages. (Reproduced with permission, open-access articles are distributed under the terms of the Creative Commons Attribution License (CC BY). Copyright © 2023 Frontiers Media S.A.). B) Photosensitive functional chitosan backbone to assemble a photosensitive polymeric nanocarrier for intracellular administration of therapeutic principles. (Reproduced with permission. Copyright © 2023 Springer Nature Limited.) C) Micromotor-immobilized enzymes to improve their long-term stability and reusability by protecting them from UV light irradiation and accelerating substrate degradation. (Reproduced with permission. Copyright © 1996-2023 MDPI.) 

Apart from synthetic polymers, natural polymers are common raw materials when encapsulating drugs. Although modulation of their properties is a little more challenging than their synthetic counterparts, they are amenable to cost-effective derivatization. Derivatization aims to introduce functionalities for coupling with other polymers, ligands, and particles or becoming susceptible to external stimuli, including temperature, pH, media, and light (^{Becerra et al., 2022}).

Functionalization can be achieved on the polymer pre- or post-assembling the nano-carriers using the proper linking chemistry (^{Fernández & Orozco, 2021}). For example, we introduced a photosensitive functionality into a chitosan backbone to assemble a photosensitive polymeric nanocarrier for intracellular administration of therapeutic principles (Figure 5B) (^{Mena-Giraldo et al., 2020}). Ultraviolet (UV) photosensitive nanocarriers were then functionalized with transmembrane peptides (TPs) for intra-cellular and site-specific drug delivery. The resulting nanobioconjugate encapsulated Nile red and dofetilide, the gold standard against auricular fibrosis, with high efficiency and biocompatibility. Furthermore, the cargo was released by exposing it to UV for a few seconds without phototoxicity. Additionally, fluorescence microscopy experiments showed that the nanobioconjugate had a higher affinity for the target cells evidenced by a higher extent of cell uptake concerning bare nanobioconjugates after 8 h of incubation. Overall, the results demonstrated that administering active principles in a spatiotemporally controlled manner through functional nanocarriers is a strategy to improve the effective ness of therapeutic regimens, with the potential to reduce their side effects.

Taking advantage of the chitosan backbone derivatized with a photostimulable molecule, we proved the concept of immobilizing enzymes into micromotors to improve their long-term stability and reusability by protecting them from UV light irradiation and accelerating substrate degradation (Figure 5C) (^{Mena-Giraldo & Orozco, 2022}). Micromotors convert different kinds of energy into movement, enhancing the kinetics and thermodynamics of the involved reactions. The movement of micromotors made of photosensitive polymers speeds up the tasks they were designed for, adding value to the inherent advantages of these naturally existing macromolecules, apart from photo stimulation. In this context, we reported on a photosensitive polymeric Janus micromotor (JM) for UV-light protection of enzymatic activity and accelerated motion-degradation of substrates. The UV-photosensitive modified chitosan JMs co-encapsulated fluorescent labeled proteins, enzymes, magnetite, and platinum nanoparticles for magnetic and catalytic motion. The JMs protected the enzymatic activity and accelerated the enzyme-substrate degradation by magnetic/catalytic propulsion. Protein-immobilized photosensitive JMs offer the potential to improve the enzyme's stability and substrate degradation efficiency.