# Highly Sensitive Detection of Glucose in the Presence of Serum Based on Signal Amplification of Persistent Luminescence Nanoparticles Functionalized by Glucose Oxidase

Zied Ferjaoui, Celina Matuszewska, Jianhua Liu, Yohann Corvis, Corinne Chanéac, Bruno Viana, Ferdaous Ben Romdhane, Daniel Scherman, Nathalie Mignet, Cyrille Richard

► To cite this version:

Zied Ferjaoui, Celina Matuszewska, Jianhua Liu, Yohann Corvis, Corinne Chanéac, et al. Highly Sensitive Detection of Glucose in the Presence of Serum Based on Signal Amplification of Persistent Luminescence Nanoparticles Functionalized by Glucose Oxidase. Advanced Optical Materials, 2025, 13 (4), 10.1002/adom.202402373. hal-05329119

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# Highly Sensitive Detection of Glucose in the Presence of Serum Based on Signal Amplification of Persistent Luminescence Nanoparticles Functionalized by Glucose Oxidase

Zied Ferjaoui, Celina Matuszewska, Jianhua Liu, Yohann Corvis, Corinne Chanéac, Bruno Viana, Ferdaous Ben Romdhane, Daniel Scherman, Nathalie Mignet, and Cyrille Richard*

A new method is presented for the in vitro detection of glucose using glucose oxidase (GOx) covalently linked to persistent luminescent nanoparticles (PLNPs). This method ensures both sensitive and specific glucose detection by exploiting the enhanced luminescence of PLNPs in the presence of H₂O₂, generated by an enzymatic reaction. To this end, three different PLNPs composed of ZnGa₂O₄:Cr³⁺ (ZGO) nanoparticles are prepared by hydrothermal synthesis at 120 °C for 6 h (ZGO1), 12 h (ZGO2), and 24 h (ZGO3), followed by a calcination at 500 °C, resulting in nanoparticles with an average hydrodynamic diameter of 100 nm ± 5 nm after grinding and centrifugation. These nanoparticles are efficiently covalently functionalized with GOx, via a PEG linker. Following the production of H₂O₂ by the enzymatic reaction between GOx bound to the ZGO surface and glucose present in 100-fold diluted serum, a significant increase in the persistent luminescent signal is observed. This phenomenon is most pronounced for ZGO2, for which a detection limit of 0.01 µM and a detection range from 0.05 to 1 µM is obtained. These results demonstrate the innovative potential of this new technique in glucose monitoring, opening up new avenues for real-time monitoring and effective management of diabetes.

## 1. Introduction

In vitro biodetection, an analytical method for detecting the presence and concentration of specific substances in a biological sample outside the living body provides essential precision and sensitivity for diagnosing various medical conditions, monitoring biomarker levels, and controlling biological processes.[1,2] A critical application of this method is glucose biodetection in diabetes mellitus, a disease characterized by high blood glucose levels that can lead to severe complications if not adequately controlled.[3,4] With 425 million adults affected by diabetes and a predicted 629 million by 2045, reliable methods for monitoring blood glucose levels are crucial.[3] They often rely on measuring devices, such as glucometers, which use specific enzymes, such as glucose oxidase (GOx), to catalyze the reaction between glucose in the sample and oxygen to produce a detectable signal, usually electrochemical or optical.[5,6]

Z. Ferjaoui, J. Liu, Y. Corvis, D. Scherman, N. Mignet, C. Richard
Université Paris Cité
CNRS
INSERM
UTCBS
Unité de Technologies Chimiques et Biologiques pour la Santé
Paris F-75006, France
E-mail: cyrille.richard@u-paris.fr

C. Matuszewska, C. Chanéac
Sorbonne Université
CNRS
Collège de France
Laboratoire de Chimie de la Matière Condensée de Paris (LCMCP)
Paris 75005, France
B. Viana
Université PSL
CNRS
IRCP
Chimie ParisTech
Paris 75005, France
F. B. Romdhane
Sorbonne Université
CNRS
Fédération de Chimie et Matériaux de Paris-Centre (FCMat)
Paris 75005, France

<figure><img src="image_3.png" /></figure>

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adom.202402373

© 2025 The Author(s). Advanced Optical Materials published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

DOI: 10.1002/adom.202402373
There are several methods of glucose detection, each with its advantages and limitations.[7,8] The electrochemical approaches commonly used in portable glucometers measure changes in current or potential in the presence of glucose but can suffer from chemical interferences and require regular calibration.[9,10] Colorimetric methods are simple and inexpensive, detecting color changes due to the reaction of glucose with specific reagents, but have limited sensitivity.[11,12] Fluorogenic techniques, based on probes that fluoresce on contact with glucose, are highly sensitive and specific but can be expensive and sensitive to photodegradation.[13,14] Finally, enzymatic methods exploit the specificity of enzymes such as GOx, which reacts with glucose to produce a detectable signal.[15,16]

Among the possible alternative methods, the use of persistent luminescence nanoparticles (PLNPs) could offer distinct advantages.[17–19] These nanoparticles represent an innovative class of materials offering significant advantages for biomedical detection, particularly due to their ability to emit light persistently after excitation by an external source.[20,21] Unlike conventional luminescent materials, which require continuous illumination to produce a detectable signal, PLNPs maintain their luminescence even after the excitation source is removed.[22] This unique property minimizes optical background noise, which is often caused by the excitation source in traditional methods, thereby enhancing the signal-to-noise ratio and enabling more accurate detection.[23,24] Furthermore, the persistent luminescent signal (PLS) of PLNPs can be precisely engineered to emit at specific wavelengths.[25] This wavelength specificity is critical in complex environments where multiple optical signals may coexist, as it allows for clear differentiation of the target signal from potential interferences caused by overlapping emissions.[25] These characteristics make PLNPs particularly suitable for biomedical applications requiring high sensitivity and specificity, such as in vivo imaging, targeted diagnostics, and real-time biosensing.[3] Another major advantage of PLNPs lies in their ability to enable prolonged detection periods without the need for continuous illumination.[24] This not only enhances their efficiency but also reduces the risks of photobleaching and phototoxicity, which are common limitations of traditional luminescent materials in biological systems.[24] These features, combined with their tunability and durable emission, position PLNPs as a powerful tool for advancing imaging techniques, improving diagnostic precision, and developing innovative sensing platforms tailored to complex biomedical environments.[26]

A recent study led by J. Li et al.[27] reported the successful synthesis of torpedo-shaped PLNPs composed of Zn₂GeO₄:Mn²⁺, Pr³⁺ (ZGMP) via a simple one-pot hydrothermal (HT) method. These pH-sensitive nanoparticles were integrated into autofluorescence-free biosensors for various analytes, paving the way for new applications in autofluorescence-free biodetection and high-level information encryption. In the context of biodetection, these authors used the GOx enzyme adsorbed on a 96-well plate and ZGMP nanoparticles dispersed in the wells to access the concentration of glucose without autofluorescence interference, with detection values as low as 0.2 µg L⁻¹ and a broader linear range compared to some previous methods. Furthermore, this method was successfully applied to glucose determination in serum samples, thus offering promising prospects for improved diabetes management.

Using a different strategy, a recent study conducted by our team, developed by J. Liu et al.,[28] reported the unexpected effect of hydrogen peroxide (H₂O₂) on the signal intensity of ZnGa₂O₄:Cr³⁺ (ZGO) nanoparticles. These ZGO NPs were prepared by an HT method at 120 °C for 24 h, then calcined at 500 °C for 5 h. In the presence of H₂O₂, an enhancement of the ZGO signal intensity was observed. This signal enhancement was used to detect and quantify H₂O₂ in various media using non-functionalized ZGO nanoparticles. As this molecule can be produced by GOx when reacting with glucose,[28] increasing amounts of glucose were incubated with GOx and ZGO for 1 h. The signal amplification of ZGO increased with the amount of glucose, and a linear relationship was obtained when the glucose concentration was between 62.5 and 250 µM, which could be fitted as a linear function, allowing the determination of the limit of detection (LOD) of glucose, which was equal to 36 µM. This unique property, namely the enhancement of the persistent luminescence signal induced by H₂O₂, represents a new way of detecting biomolecules and opens the door to numerous applications in the field of biological assays. In the same context, a recent study developed by L. Wang et al.[29] introduces nanoparticles of ZnGaSnO₄ doped with Cr³⁺ and Y³⁺ (ZGSCY) coated with a mesoporous organosilica layer (ZGSCY@MON). Using these nanoparticles synthesized by the combustion method, Wang et al. also observed an increase of their luminescence in the presence of H₂O₂, confirming on a similar composition, the signal enhancement first reported with ZGO in 2023.[28] The ZGSCY@MON nanoparticles have demonstrated their ability to detect glucose with high sensitivity and specificity in human serum. A detection limit of 0.21 µM was achieved, with a detection range from 1 to 100 µM. This method also provides an effective approach for the detection of glucose without autofluorescence in human serum, using ZGSCY and GOx dispersed in the serum.

Following on from our initial work, we then reported the effect of both the method used to prepare ZGO NPs (HT at 120 °C for 24 h followed by calcination at 500 or at 750 °C), and the size of the nanoparticles (60, 100, and 180 nm) on the detection limit of H₂O₂ and glucose in serum. Such optimizations of the nanoparticle synthesis parameters have resulted in detection limits of ≈2.1 µM for H₂O₂ and 0.13 µM for glucose in HS.[30]

In the present work, we explored an alternative strategy for the detection of glucose, based on the covalent grafting of GOx onto the surface of the ZGO NPs, previously prepared at 120 °C for 24 h (Scheme S1, Supporting Information). In addition, two other ZGO nanoparticles were synthesized using the same HT method at a temperature of 120 °C but for 6 h or for 12 h, respectively. To recover the nanoparticles, each pristine powder obtained after sintering at 500 °C was ground for one hour using a mechanical mill and centrifuged at 4600 rpm for 10 min. Finally, to covalently link GOx enzymes onto the ZGOs, each type of nanoparticle was first functionalized with APTES, then with NHS-PEG-biotin, and finally with avidin-glucose oxidase enzyme (avidin-GOx). Of the three nanoparticles prepared, those synthesized at 120 °C for 12 h (ZGO2-GOx), have a LOD of ≈0.005 µM in PBS buffer, demonstrating a 4-log improvement in sensitivity compared with the non-functionalized particles reported by our team in the original study.[28] In addition, a tenfold improvement in the detection limit (0.01 µM) was obtained with ZGO2-GOx in serum compared to results we obtained previously
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Schematic illustration of the synthesis of ZGO nanoparticles.</figcaption></figure>

using the ZGO NPs non-covalently functionalized with GOx.[29]
Our results are also 20-fold better than the LOD obtained in
the previously mentioned work published by Wang et al. using
ZGSCY@MON.[29] Our work highlights the potential of func-
tionalized ZGO nanoparticles in the field of biomedical detec-
tion field. The results indicate that through synthesis and func-
tionalization, it is possible to optimize nanoparticle properties for
specific and sensitive glucose detection. This advance could have
a significant impact on diabetes monitoring and management,
enabling accurate and reliable glucose measurement under real-
world conditions, opening avenues for new clinical applications,
and improving the quality of life of diabetic patients.

**2. Experimental Section**

**2.1. Materials**

Zinc nitrate hexahydrate (>99%), gallium oxide (99.999%), and chromium (III) nitrate nonahydrate (99.9%) were obtained from Alfa Aesar, Karlsruhe, Germany. The ammonia solution (30 wt%) and hydrochloric acid were sourced from Carlo Erba. 3-Aminopropyl-triethoxysilane (APTES) (99%), Dimethylformamide (>99.9%) (DMF), Nitric acid (70 wt%), Phosphate Buffered Saline (PBS), Human Serum, Glucose Oxidase, and Glucose (>99.5%) were obtained from Sigma-Aldrich. Biotin-PEG-NHS (MW: 3145 g mol⁻¹) was from Iris Biotech GmbH. Avidin-glucose oxidase was supplied by Rockland. A commercial glucose assay kit was obtained from Abcam (ab65333).

**2.2. Preparation of ZGO Nanoparticles**

The ZGO nanoparticles were synthesized according to a well-
defined protocol.[31-33] First, 1.675 g of gallium oxide (Ga₂O₃)
(8.94 mmol) was dispersed in 10 mL of 35% nitric acid (HNO₃).
This mixture was subsequently transferred to a Teflon-coated

stainless-steel autoclave and heated to 150 °C for a duration of
four days until complete dissolution of the gallium oxide. Sec-
ond, a solution containing 0.016 g of Cr(NO₃)₃·9H₂O (0.04 mmol)
and 2.668 g of Zn(NO₃)₂·6H₂O (8.97 mmol) dissolved in 10 mL
of demineralized water was prepared. This solution was mixed
with the previously obtained Ga(NO₃)₃ solution while maintain-
ing continuous agitation. The pH of the solution was adjusted
to 7.5 by adding ammonia (≈8 mL) drop by drop. Subsequently,
the resulting solution was stirred for 3 h at room temperature and
then transferred to a Teflon-coated stainless-steel autoclave. Heat-
ing was carried out at 120 °C for either 6, 12, or 24 h (Figure 1).
Once the heating process was completed, the resulting product
was thoroughly washed with water and ethanol using centrifuga-
tion at 4600 rpm for 10 min, then dried at 60 °C for 1 h. The result-
ing white powder was subjected to a calcination process at 500 °C
for 5 h. To obtain ZGO particles of nanometric size, 500 mg of
powder were ground for one hour using a mechanical grinder
in the presence of 500 µL of a 5 mm hydrochloric acid (HCl) so-
lution. Subsequently, the solution was transferred to 50 mL of
HCl, and the suspension was stirred overnight to recover ZGO
nanoparticles.

Finally, centrifugations were performed at 4600 rpm for
10 min to obtain a monodisperse particle suspension.

**2.3. Preparation of Biotin-Functionalized ZGO NPs**

To prepare ZGO-NH₂ nanoparticles, 1% by weight of APTES was
added to a suspension of ZGO-OH particles at a concentration of
2.5 mg mL⁻¹ in 4 mL of DMF. The mixture was sonicated for
the first 2 min using a Branson 1210 ultrasonic cleaner and then
vigorously stirred at room temperature for 6 h. To remove excess
APTES, the nanoparticles were washed three times with DMF.[31]
Next, for functionalizing the ZGO-NH₂ nanoparticles with NHS-
PEG-Biotin, 60 mg of NHS-PEG-biotin was added to a suspen-
sion of 10 mg of ZGO-NH₂ dispersed in 4 mL of DMF.[34] The
mixture was sonicated for 1 min and stirred overnight at 50 °C in
an oil bath. Following this, the excess NHS-PEG-Biotin was removed by washing the nanoparticles three times with DMF and using centrifugation to separate the functionalized particles from unreacted polymer. The functionalized nanoparticles were then stored at 4 °C until use.

## 2.4. Preparation of ZGO-GOx NPs

One milligram of ZGO-PEG-biotin was thoroughly washed multiple times with a PBST solution, a mixture comprising PBS and 0.05% Tween 20, to ensure proper cleaning and removal of any impurities. Subsequently, the ZGO-PEG-biotin was dispersed in 1 mL of PBST, and 100 µg of avidin-GOx was introduced into the suspension. The mixture was left at room temperature for 1 h, allowing for the binding of avidin-GOx to the ZGO-PEG-biotin nanoparticles. The resulting ZGO-GOx nanoparticles, underwent further washing steps (centrifugations at 14 500 rpm for 15 min) with PBST to eliminate any excess or unbound avidin.

## 2.5. NPs Characterization Techniques

For the thermogravimetric analyses (TGA), a supersonic thermobalance SETSYS (SETARAM) was used. Dry samples of ≈5 mg were placed in an alumina crucible. The samples were heated from room temperature to 800 °C at a heating rate of 5 °C min⁻¹ under an argon atmosphere. The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the nanoparticles were characterized using DLS conducted with a Zetasizer Nano ZS instrument (Malvern Instruments, Southborough, MA, USA). The instrument was equipped with a helium-neon laser operating at 632.8 nm and a power of 5 mW, with a detection angle of 173°. Transmission Electron Microscopy (TEM) operating at 120 kV was employed to characterize the size, shape, and crystalline structure of the ZGO nanoparticles. High-resolution TEM (HRTEM) and high-angle annular dark-field scanning transmission electron microscopy analyses were performed on a TEM equipped with energy-dispersive X-ray spectroscopy (EDS) analysis system to gather detailed information.

The stability of the synthesized nanoparticles was evaluated at a pH of 7.4 and a temperature of 37 °C. To do this, a suspension containing 0.5 mL of ZGO-GOx nanoparticles (at a concentration of 0.1 mg mL⁻¹) dispersed in human serum diluted 100 times at the desired pH was prepared and maintained at rest in a quartz cuvette at 37 °C for 15, 30, 45, and 60 min. The intensity of the dispersion was periodically recorded using DLS and compared to that of the freshly prepared and initially measured sample.

## 2.6. Bradford Assays on ZGO-GOx

For protein quantification, avidin-GOx bound to ZGO was assessed using the Bradford assay. The standard concentration range utilized was from 0 to 25 µg mL⁻¹, with avidin-GOx serving as the protein standard. To establish the standard curve, 150 µL of varying concentrations of avidin were added to microplate wells, followed by the addition of 150 µL of Coomassie reagent. The standard curve was generated after a 10-minute incubation at

room temperature, with absorbance measured at 560 nm. Absorbance readings were recorded based on the concentration of the protein standard. To quantify the protein on ZGO, 150 µL of ZGO-GOx (0.1 mg mL⁻¹) was added to the plate. Following the addition of 150 µL of Coomassie reagent, protein concentration on the plate was assessed based on absorbance readings and the previously established standard curve. The protein standard was prepared using the same amount of ZGO-OH NPs to correct for the effects of PLNP absorbance in the conducted measurements.

## 2.7. H₂O₂ Impact on ZGO-GOx PLS

The effect of H₂O₂, produced by the enzymatic reaction between ZGO-GOx and added glucose, on the persistent luminescence signal of ZGO nanoparticles was evaluated using ZGO-GOx nanoparticles synthesized under the three distinct conditions (6, 12, and 24 h). Initially, the ZGO-GOx nanoparticles were washed with distilled water and then centrifuged at 14 g for 20 min to recover them in the pellet. Subsequently, 50 µL of ZGO-GOx suspensions in PBS at concentrations of 0.05, 0.1, and 0.2 mg mL⁻¹ were prepared and added in triplicate to a 96-well plate. Then, 50 µL of glucose at different concentrations from 0 mM to 500 µM were prepared in PBS with varying percentages of human serum, namely 0.1%, 1%, and 10%, and added to the ZGO-GOx aqueous suspension. The resulting mixture, reaching a final volume of 100 µL, was incubated for 15, 30, 45, or 60 min at 37 °C. Persistent luminescence signal decays were collected over 5 min using a photon counting device (Biospace Optima camera) after UV excitation at 254 nm for one minute. Subsequently, these data were analyzed using M3 Vision software to evaluate the response of ZGO-GOx nanoparticles to different glucose concentrations. This methodology allowed for the assessment of the sensitivity of ZGO nanoparticles to varying glucose concentrations, which was essential for characterizing their performance in the detection of this compound.

## 2.8. Validation of Glucose Detection with ZGO-GOx Nanoparticles

Glucose detection was performed by incubating 50 µL of ZGO-GOx (0.1 mg mL⁻¹) and 50 µL of glucose at various concentrations (0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 125, 250, and 500 µM), prepared in PBS or human serum diluted 100-fold, in a microplate at 37 °C for 15, 30, 45, and 60 min to generate H₂O₂. Following each incubation period, signal enhancement was measured and analyzed as described previously.[27] The proposed method underwent validation according to standards established by internationally recognized publications,[35,36] covering aspects such as linearity, limits of detection and quantification (LOD and LOQ), selectivity, and precision. Linearity was assessed by constructing graphs of enhancement ratios against glucose concentrations, ranging from 0.05 to 500 µM. Sensitivity was determined using the LOD (3 SD/S) and the LOQ (10 SD/S), based on the standard deviation of different blank samples (SD) and the slope of the calibration curve (S). The calibration curves were determined by plotting the amplifications after subtracting the amplification of the control (representing zero glucose concentration) against
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Schematic representation of the different steps for ZGO functionalization by PEG-Biotin and avidin-GOx.</figcaption></figure>

the glucose concentration to perform linear fitting. Method selec-
tivity and specificity were evaluated by comparing the measured
concentrations to the effectively added glucose concentration ac-
cording to the formula (Recovery rate (%) = (Measured quan-
tity/Added quantity) × 100). Finally, precision was examined in
terms of repeatability, assessed by conducting three analyses on
five different concentrations for each analyte, and reproducibility
was evaluated over a period of three consecutive days.

**2.9. Statistical Analysis**

The analysis involved presenting values as the mean and stan-
dard deviation derived from a minimum of three experiments.
To assess the significance between experimental groups, an
unpaired Student's t-test (unless otherwise specified) was con-
ducted, with a *p*-value < 0.05 was considered statistically signifi-
cant.

**3. Results and Discussion**

The three types of ZGO were synthesized via HT treatment at
120 °C for varying heating durations: 6 h (ZGO1), 12 h (ZGO2),

and 24 h (ZGO3) followed by calcination at 500 °C for 5 h. These
ZGO variants were isolated after crushing and centrifugation at
4600 rpm for 10 min. These nanoparticles were first function-
alized with APTES to yield ZGO-NH₂, which then allows func-
tionalization with NHS-PEG-biotin to produce ZGO-PEG-Biotin,
and finally modified with avidin-GOx, resulting in ZGO-GOx
(Figure 2). To confirm the presence of the organic layer and to
optimize the amount of PEG-biotin grafted onto the ZGO, TGA
was carried out. The results show a weight loss percentage of
≈6% after functionalization with APTES (Figure S1, Supporting
Information). To optimize the grafting density with PEG-biotin,
two different temperatures were tested: room temperature (R.T.)
and 55 °C. As shown in Figure S1 (Supporting Information), the
weight loss percentage of ZGO-PEG-biotin was ≈8.5% for NPs
functionalized at R.T. and ≈12% when functionalized at 55 °C,
indicating that more PEG-biotin can be bound at 55 °C. From
these data, we can estimate the grafting density. For PEG-biotin,
it is estimated to be ≈28 nmol mg⁻¹ at R.T. and 43 nmol mg⁻¹
at 55 °C. The PEG-Biotin was then grafted at 55 °C onto
the ZGO.

Hydrodynamic diameter, PDI, and zeta potential measure-
ments were performed using DLS after each chemical modifica-
tion step. **Figure 3a,b** illustrates the progressive increase in ZGO
particle size through the synthesis stages: starting at 100 (±5) nm
<figure><img src="image_6.png" /><figcaption>**Figure 3.** a) Analysis of nanoparticle hydrodynamic diameter, b) PDI, c) Zeta potential obtained using DLS, and d) The amount of avidin grafted on the surface of ZGO. Results represent average ± standard deviation. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.</figcaption></figure>

with a PDI of 0.141 for ZGO-OH, it increases to 155 (±10) nm with a PDI of 0.119 for ZGO-NH₂, to 180 (±7) nm with a PDI of 0.135 ± 0.024 for ZGO-PEG, and finally reaches 210 (±5) nm with a PDI of 0.198 ± 0.019 for ZGO-GOx. Figure 3c displays the zeta potential evolution of ZGO, starting at 30 (±5) mV for ZGO-OH, it reduces to 7 (±2) mV for ZGO-biotin and stabilizes at 5 (±2) mV for ZGO-avidin-GOx.

The observed changes in hydrodynamic diameter and zeta potential of ZGO during their functionalization confirm the success of this process.[37,38] The gradual increase in size and adjustments in PDI attest to the effective addition of various functional groups and avidin-GOx in a core-corona structure. Similarly, variations in zeta potential indicate significant changes in surface charge, crucial for confirming the attachment of each molecule. These findings underscore the successful functionalization at each step.

To confirm and quantify the amount of avidin-GOx grafted onto the surface of ZGO, the Bradford assay was utilized.[39] This allowed for the evaluation of avidin-GOx presence on the ZGO surface. The standard calibration curves of avidin-GOx are depicted in Figure S2 (Supporting Information). Based on this calibration curve ($Y = 0.03X + 0.02$) ($R^2 = 0.99$), the amount of avidin-GOx on the ZGO surface was estimated to be 60 (±6) µg mg⁻¹ of ZGO (Figure 3 d). An estimation of the amount of protein per nanoparticle is presented in Table S4, Supporting Information). These results once again confirm the successful functionalization of ZGO with avidin-GOx, demonstrating the effectiveness of our surface modification strategy for the specific anchoring of active biomolecules.

The FTIR spectrum of ZGO nanoparticles, shown in Figure S3 (Supporting Information), exhibits two characteristic bands corresponding to the vibrations of the metal-anion Zn–O and Ga–O
bonds, located in the range of 650 to 800 cm⁻¹. After functionalization with GOx, the spectrum displays bands similar to those observed for GOx alone, as well as a broad and intense band ≈1639 cm⁻¹, attributed to the C=O bond vibration in the amide group.[⁴⁰] Additionally, a band ≈3300 cm⁻¹, associated with the vibration of amine groups, is also present.[⁴¹,⁴²] These observations confirm the successful coating of GOx on the ZGO NPs, thus validating the functionalization of the ZGO surface.

To assess the core size, morphology, and crystal structure of ZGO-GOx nanoparticles, we have analyzed ZGO2-OH and ZGO2-GOx using transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HR-TEM), and elemental mapping analysis via scanning transmission electron microscopy energy dispersive X-ray spectroscopy.

The TEM images, illustrated in **Figure 4a**, display spherical nanocrystals characterized by a relatively uniform size distribution and good interparticle separation, observable both before and after the functionalization process. This uniformity emphasizes that the functionalization process does not significantly affect the primary characteristics of the particles. The analysis of size distributions for ZGO2-OH reveals an average of ≈9.5 nm, compared to a slightly higher average, ≈13 nm, for ZGO2-GOx (Figure 4b) (calculated from 400 randomly selected particles). The size difference might be due to numerous centrifugation steps during the preparation of ZGO2-GOx, which thus, could result in the loss of some of the smallest nanoparticles and as a consequence the shift of the particle size distribution.

Moreover, the HR-TEM images (Figure 4a, inset and Figure S4, Supporting Information) clearly illustrate the presence of multiple well-defined crystalline planes, which are consistent with the characteristic spinel structure of ZGO2-GOx. This confirms the high crystalline quality of the nanomaterial. Indeed, the measured distances between the diffraction fringes, namely 4.81, 2.95, and 2.52 Å, correspond to the interplanar spacings, denoted as “d”, of the crystalline planes (111), (220), and (311) typical of the spinel structure of zinc gallate.[⁴³,⁴⁴] This correlation is based on the geometry of the spinel crystalline structure, where each set of crystalline planes presents characteristic spacings that are identifiable and comparable to the theoretical values provided by crystallography (referenced in the JCPDS No. 38–1240),[⁴⁴]

Additionally, the elemental mappings presented in Figure 4c indicate that ZGO2-GOx primarily contains elements such as zinc (Zn), gallium (Ga), and oxygen (O), and chromium (Cr), aligning with its anticipated chemical composition. The presence of silicon (Si) confirms the effectiveness of the functionalization with APTES on the surface of ZGO2-GOx.

We also performed X-ray diffraction (XRD) characterization of the pristine samples, as well as the ZGO sample after surface modifications (Figure S5, Supporting Information). The XRD patterns recorded for the pristine ZGO samples (without surface modifications) closely match the pattern of cubic ZnGa₂O₄ (PDF 038–1240, Fm-3m space group),[⁴⁴] confirming the purity of the samples. Additionally, no significant changes were observed in the XRD pattern of the sample after surface modifications (ZGO2-GOx). The average crystallite size was calculated from the broadening of the XRD peaks using the Scherrer equation. The average crystallite sizes for ZGO-1, ZGO-2, and ZGO-3 were determined to be 11.1, 8.9, and 12 nm, respectively, which are consistent with the size distributions obtained from TEM.

### 3.1. Enhancement of PLS of ZGO-GOx in Presence of H₂O₂

In this section, we assessed the optical properties, specifically the emitted PLS by ZGO1-GOx, ZGO2-GOx, and ZGO3-GOx following their excitation under UV at 254 nm for 1 min, at a concentration of 0.1 mg mL⁻¹. The results, depicted in Figure S6 (Supporting Information), demonstrate that ZGO3, synthesized at 120 °C for 24 h, emits a more intense PLS compared to ZGO1 and ZGO2, which were prepared at 120 °C for 6 and 12 h, respectively. This observation highlights the significant impact of the duration of hydrothermal treatment on the optical properties of ZGO composites, a phenomenon already corroborated by other published studies.[¹⁷] The higher signal for ZGO3-GOx might be attributed to prolonged HT treatment, improving the structure and crystallinity of ZGO, thereby influencing its electronic properties and, consequently, its optical behavior.

Subsequently, we explored the impact of H₂O₂, produced by the enzymatic reaction between GOx grafted onto ZGO-GOx using glucose at a concentration of 500 µM (Figure 5a). Glucose detection based on PLS amplification relies on the reaction between glucose and the GOx immobilized on NPs (Figure 5a). During the oxidation of glucose, GOx catalyzes the following reaction: $C_6H_{12}O_6 + O_2 \rightarrow C_6H_{12}O_7 + H_2O_2$. This analysis was conducted by varying several parameters, including the concentration of ZGO-GOx, the incubation time at 37 °C, and the detection duration. Figure 5b revealed significant variations in signal amplification rates based on the particle concentration. Notably, ZGO2-GOx particles showed a marked PLS amplification at a concentration of 0.1 mg mL⁻¹, compared to ZGO1-GOx and ZGO3-GOx at the same concentration. However, at concentrations of 0.05 or 0.2 mg mL⁻¹, this difference becomes insignificant, and the PLS amplification is not as pronounced as that achieved with a concentration of ≈0.1 mg mL⁻¹. Consequently, we set the particle concentration to 0.1 mg mL⁻¹ for subsequent analyses.

Figure 5c highlights the effect of incubation time on PLS amplification, focusing on the dynamics of the reaction between grafted GOx on ZGO and glucose. It was observed that ZGO2-GOx shows a significant signal increase after 30 min of incubation compared to 15 min, with no notable difference for longer periods of 45 and 60 min. For ZGO1-GOx, a significant increase is noted between 15, 30, and 45 min of incubation, but the difference is not significant between 45 and 60 min, indicating that 45 min is the optimal incubation duration for this sample. Similarly, ZGO3-GOx exhibits a significant signal improvement between 15 and 30 min, with no notable variation for incubations of 30, 45, and 60 min, suggesting that ≈30 min of incubation is sufficient for a notable PLS amplification.

Finally, the influence of human serum on the PLS amplification of ZGO-GOx was studied by adjusting the serum percentage to 0.1%, 1%, and 10%, while adding a constant glucose concentration of 500 µM. Figure 5d indicates a reduced amplification at 10% serum for all samples, whereas a significant amplification is observed at 1% and 0.1% serum for all ZGO-GOx.

These findings suggest that the presence of human serum can modify the reactivity and efficiency of the interaction between ZGO-GOx and glucose, likely due to the serum's complex protein content, which may interfere with GOx activity or with the interaction between the produced H₂O₂ and ZGO-GOx. At
<figure><img src="image_7.png" /><figcaption>**Figure 4.** a) TEM images of ZGO2-OH and ZGO2-GOx; inset shows an HR-TEM image of ZGO2-GOx with a scale bar of 10 nm. b) Size distribution of ZGO2-OH and ZGO2-GOx. c) Elemental mapping images of ZGO2-GOx.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 5.** a) Principle of glucose detection based on PLS enhancement. Influence of ZGO-GOx concentration (b), incubation time (c), and human serum dilution (d) on the enhancement of signal amplification after contact with 500 µM glucose. Results represent the mean ± standard deviation (experiments conducted in triplicate, n = 3). \*p < 0.05, \*\*p < 0.01.</figcaption></figure>

higher serum concentrations (10%), proteins and other components may bind to the particle surface, limiting glucose access to GOx or the diffusion of H₂O₂ toward the active sites of ZGO, thereby reducing signal amplification.[45] Conversely, at lower serum concentrations (0.1% and 1%), the inhibitory effect is diminished, allowing for a more effective interaction between glucose and GOx, as well as a more efficient diffusion of produced H₂O₂ toward ZGO, leading to a significant PLS amplification. This analysis highlights the importance of the medium's composition on the performance of ZGO-GOx-based detection systems, underscoring the need to optimize the serum percentage to ensure maximum sensitivity and reliability of measurements in complex biological samples.

This study highlights the significance of experimental conditions, such as synthesis time, particle concentration, incubation time, and the presence of biological components like serum, in PLS amplification of ZGO-GOx composites in the presence of H₂O₂. It is demonstrated that the duration of hydrothermal

treatment directly affects the optical properties of ZGO, with increased signal amplification observed for samples treated for 12 h.[46] Similarly, optimizing the concentration of ZGO-GOx and the incubation time proves crucial for maximizing the signal response during the detection of H₂O₂ generated by the enzymatic reaction between GOx and glucose. Furthermore, the study demonstrates the significant impact of human serum on the interaction between the produced H₂O₂ and the surface of the nanoparticles, highlighting the complexity of using the ZGO-GOx system in undiluted real biological environments. However, from the point of view of real-life application, given the mM concentration of glucose in the blood and the submicromolar sensitivity of our technique, the serum will have to be diluted by more than 100 times for routine glucose assay, ensuring optimal PLS amplification response.

The present findings provide valuable insights for the enhancement of enzyme-based sensors utilizing ZGO-GOx, offering promising prospects for the development of low-cost and
<figure><img src="image_9.png" /><figcaption>**Figure 6.** Hydrodynamic diameter (a) and PDI (b) of ZGO-GOx NPs dispersed in human serum at pH 7.4 and 37 °C as a function of time. Experiments were performed in triplicate, *n* = 3.</figcaption></figure>

more sensitive and specific detection technologies, suited for
complex biomedical applications.

## 3.2. Stability of ZGO-GOx in Human Serum at 37 °C

The ZGO-GOx, at a concentration of 0.1 mg mL⁻¹, were incubated in 1% human serum at 37 °C for durations of 15, 30, 45, and 60 min. After each incubation period, the hydrodynamic diameter (Figure 6a) and PDI (Figure 6b) of the ZGO-GOx were measured using DLS at 15-minute intervals for one hour. The stability results are presented in Figure 6, and show, taking into account the error bars, that there are no significant variations in either the diameters or the PDI of the ZGO-GOx systems over the observation period. This stability in the measurements of hydrodynamic diameter and PDI indicates that the ZGO-GOx nanoparticles maintain their size and uniform distribution in a medium containing 1% human serum at 37 °C, for at least an hour. This observation is crucial as it reveals that the ZGO-GOx preserve their physical characteristics under conditions that take into account the biological environment, a key aspect for future biomedical applications, including the in vitro biodetection of glucose in serum after a 100-fold dilution.[47,48]

## 3.3. Physicochemical Stability and Catalytic Activity of ZGO-GOx during Prolonged Storage at 4 °C

The ZGO-GOx NPs, at a concentration of 0.1 mg mL⁻¹, were stored in PBS at 4 °C and pH 7 for periods of 7, 30, and 90 days. After each storage period, the hydrodynamic diameter, PDI, and zeta potential of the ZGO-GOx were measured using DLS. Stability results, shown in Figure S7 (Supporting Information), indicate no significant variation in diameter, which remained steady

≈230 nm ± 10 nm with a PDI between 0.1 and 0.18. Additionally, the zeta potential remained ≈7 mV ± 2 mV throughout the observation period. The consistency of hydrodynamic diameter, PDI, and zeta potential values suggests that the ZGO-GOx NPs maintain their size and uniform distribution in PBS at 4 °C for at least 90 days.

We also investigated the impact of extended storage on their detection activity. Following each storage interval, we assessed the signal amplification of ZGO-GOx upon addition of 100 mM glucose, after one hour of incubation. Results show that regardless of the storage duration, the NPs continue to exhibit signal amplification of ≈3.5 ± 0.5 (Figure S8, Supporting Information). This confirms that the catalytic activity of GOx attached to the ZGO surface remains intact, also highlighting the stability of the optical properties of ZGO-GOx over 90 days, with consistent sensitivity and signal amplification following H₂O₂ production. This study demonstrates the long-term stability of ZGO-GOx NPs in terms of size, surface charge, and catalytic activity under low-temperature storage conditions.

## 3.4. Glucose Detection in PBS and Human Serum

Under optimal conditions, the analytical performance for glucose detection was meticulously evaluated in PBS and human serum (1%), using the 0.1 mg mL⁻¹ three types of particles: ZGO1-GOx (Figure S9, Supporting Information), ZGO2-GOx (Figure 7; Figure S10, Supporting Information), and ZGO3-GOx (Figure S11, Supporting Information), each at a concentration of 0.1 mg mL⁻¹. At first, this study was motivated by the need to understand the impact of incubation time on the LOD for each type of particle, a critical variable for optimizing glucose sensors in various clinical and research contexts. Consequently, the particles were incubated at 37 °C with different glucose concentrations for
<figure><img src="image_10.png" /><figcaption>**Figure 7.** a) Persistent luminescence signal decays of ZGO2-GOx with increasing glucose concentration in PBS and human serum. In the inset: Persistent luminescence signal versus [glucose], b) enhancement ratio applied to glucose detection, and c) linear relationship between the enhancement ratio and glucose concentration in PBS and human serum. Experiments were performed in triplicate, n = 3.</figcaption></figure>
periods ranging from 15 to 60 min. The results, illustrated in **Figure 7a** and Figures S8–S11 (Supporting Information), highlight a gradual and dependent increase in the PLS emitted by the particles with the addition of glucose. This relationship not only reflects the inherent differences between the types of particles but also emphasizes the importance of incubation time and the detection environment for the overall sensitivity of the system (Table S2, Supporting Information).

The remarkable linearity between PLS amplification and glucose concentrations, ranging from 0 to 500 µM, underscores the particles' effectiveness in accurately quantifying glucose. This characteristic is crucial for the development of reliable glucose sensors, capable of providing precise measurements across a wide range of concentrations, which is particularly relevant for monitoring glucose levels under various clinical conditions.

The ZGO1-GOx demonstrated a notable capability to detect glucose concentrations as low as 0.03 µM in PBS after 30 min of incubation, whereas in 1% human serum, the lowest LOD achieved was 0.07 µM after 45 min (Figure 8; Figure S9, Supporting Information). This discrepancy between the environments highlights the impact of serum components on the biosensor performance, underlining the importance of this consideration for clinical applications. ZGO2-GOx, on the other hand, revealed exceptional sensitivity, with a LOD of 0.005 µM in PBS and 0.011 µM in 1% human serum (Figure 8). These findings suggest that the specific composition of ZGO2-GOx could offer distinct advantages for detecting low glucose concentrations. Last, ZGO3-GOx exhibited LODs of 0.072 µM in PBS and 0.130 µM in 1% human serum after 45 min of incubation, indicating less competitive sensitivity but potentially influenced by incubation conditions and interactions with the environment (Figure 8; Figure S11, Supporting Information). On the other hand, we evaluated and compared the LOD for ZGO-GOx systems, where GOx is covalently attached to the surface of ZGO, against initial results with unfunctionalized ZGO. This new method demonstrates a substantial improvement in sensitivity, being ≈7200 times (4 log) greater.[28] This increase is due, on one hand, to a more efficient production of H₂O₂ when glucose reacts with GOx near the ZGO surface, and on the other hand, to the optimization of the ZGO preparation method, which facilitates a significant amplification of the signal. This progress holds promise for enhancing the accuracy and reliability of glucose sensors, marking a significant step toward improved medical applications.

To evaluate the precision and accuracy of the assay, we measured glucose levels, the coefficient of variation (CV), and the recovery rate in two PBS and human serum samples following the addition of various glucose concentrations. The CVs for all tests were below 15%, and the recovery rates ranged from 95% to 110% (Table S3, Supporting Information), meeting the acceptance criteria for bioanalytical method validation (CV ≤ 15%; recovery between 90% and 110%).[49] These results demonstrate that the ZGO-GOx nanosystem exhibits high sensitivity and excellent reliability for glucose detection in human serum. To conclude these findings, the outstanding performance of ZGO2-GOx, which exhibited superior signal amplification compared to ZGO1-GOx and ZGO3-GOx, deserves emphasis. This efficiency is manifested by an exceptionally low LOQ in both PBS and human serum, reaching 0.016 and 0.033 µM respectively after 30 min of incubation at 37 °C. These performances are markedly

distinct from those observed during other incubation periods (15, 45, and 60 min), where LOQs are higher, regardless of the medium. This LOQ optimization after 30 min of incubation with ZGO2-GOx underscores the critical importance of temporal adjustment to enhance sensor sensitivity (Figure 8).

The accuracy in glucose measurement is further supported by exemplary linearity between signal amplification and glucose concentration, spanning ranges of 0.05 to 1 µM and 0.01 to 5 µM. The determined equations, $L/Lo = 0.59C + 0.09$ and $L/Lo = 0.33C + 0.68$, with correlation coefficients of 0.99 for both, valid in both PBS and human serum, confirm the reliability and reproducibility of the ZGO2-GOx sensors across a broad range of glucose concentrations. This ability to offer high precision and sensitivity, even for very low glucose concentrations, positions ZGO2-GOx as promising tools for glucose detection in clinical contexts, where speed and accuracy are crucial. These observations highlight the complexity of glucose detection and the importance of selecting particles and incubation conditions tailored to each specific application, paving the way for future research to refine glucose sensor technology with the ultimate goal of improving diabetic patient care through precise and real-time glucose monitoring.

To assess the specificity of the assay, we conducted a control experiment in which glucose was replaced with various biomolecules and electrolytes that could potentially interfere with the assay. These substances, commonly present in complex biological samples, included carbohydrates, proteins, and ions, tested under identical conditions. Specifically, a suspension of ZGO2-GOx at a concentration of 0.1 mg mL⁻¹ was prepared and subsequently incubated with 100 mM of each interfering substance used in this study. As shown in Figure S12 (Supporting Information), the PLS signal of ZGO2-GOx in the control groups remained negligible, in stark contrast to the PLS signal observed after incubation with glucose. This intense PLS signal in the experimental group confirmed the high selectivity and specificity of the assay, thereby validating the effectiveness of the ZGO2-GOx nanosystem for glucose detection in complex biological samples such as serum.

As illustrated in Table S4 (Supporting Information), the present nano-biosensor displays superior sensitivity with a detection limit lower than that of many traditional methods, including fluorescence, colorimetric, electrochemical, and chemiluminescence techniques for glucose measurement, as illustrated in Table S3 (Supporting Information). For instance, the study by X. Chen et al.[50] introduces an innovative technique for glycemic monitoring using upconversion nanoparticles (UCNP) and a quinone-imine complex, which reduces the fluorescence of UCNP s in the presence of glucose. This method, based on an enzymatic reaction catalyzed by GOx, allows for precise glucose detection in a range of 2 to 240 µmol L⁻¹ with a detection limit of 1.0 µmol L⁻¹. Y. Tang et al.[51] developed a glucose sensor utilizing the amplified fluorescence of silver nanoparticles (AgNPs) linked to CdSe quantum dots (QDs). The AgNP-QD complexes, formed via boronic ester bonds, exhibit fluorescence up to nine times more intense. In the presence of glucose, these complexes disassemble, resulting in a fluorescence reduction that is proportional to the glucose concentration (from 2 to 52 mM, with a detection limit of 1.86 mM). In another approach, P. Wu et al.[52] presented the conjugation of glucose oxidase with Mn-doped ZnS
<figure><img src="image_11.png" /><figcaption>**Figure 8.** LOD of glucose in PBS and serum using three types of nanoparticles, ZGO1-GOx, ZGO2-GOx, and ZGO3-GOx, after different incubation times with glucose at 37 °C: 15, 30, 45, and 60 min. Experiments were performed in triplicate, *n* = 3. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.</figcaption></figure>
quantum dots for glucose detection. This sensor relies on the quenching of QD phosphorescence by H₂O₂ produced during the enzymatic oxidation of glucose. The biosensor allows detection down to 3 µM, with two linear ranges (10 µM–0.1 mM and 0.1–1 mM). Finally, Y. Liu et al.[53] proposed a novel method for the precise detection of blood glucose, which is critical in diabetes management. This method utilizes a UCNPs-PDA (poly-dopamine) nanosystem to detect glucose in human serum and whole blood. Due to the affinity of lanthanide ions on the surface of UCNPs, dopamine forms a PDA shell that quenches the luminescence of UCNPs. This quenching can be inhibited by the H₂O₂ produced during glucose enzymatic oxidation, enabling sensitive glucose detection as low as 1.2 µM. S. Aliakbarpour et al.[54] developed a colorimetric platform for glucose detection in human serum, employing gold/silver nanocages that change color in response to H₂O₂ produced by the interaction of glucose with GOx. This method enables sensitive glucose detection between 1.0 and 50 µM, with a detection limit of 0.4 µM. S. Chatterjee et al.[55] devised an electrochemical sensor for glucose, using a graphene composite with iron (III) phthalocyanine tetrasulfonic acid and nickel nanoparticles (Gr/FePc/Ni). This sensor reveals high electrocatalytic activity and high sensitivity for glucose, detectable from 5 to 930 µM, with a detection limit of 0.227 µM. It was successfully employed to measure glucose in human blood samples, demonstrating a recovery rate of ≈99.48 to 99.94%. X. Yi Wang et al.[56] developed a glucose sensor based on electrochemiluminescence (ECL) without a co-reactant, using Ru(bpy)₃²⁺ on an electrode modified by an ionic liquid. The ECL-RET system with methylene blue, adjusted by the presence of GOx, facilitates glucose detection in a concentration range of 10 µM to 1 mM with a detection limit of 1.75 µM. We also sought to compare our results with commercially available glucose kits (purchased from Abcam). The LOD determined with this kit is ≈0.2 µM (Figure S13, Supporting Information). Therefore, we have improved this LOD by 40-fold by using our NPs. This exceptional performance, marked by increased sensitivity and the ability to detect low concentrations of glucose, underscores the effectiveness of our new proposed detection method. Achieving such a low detection limit can be attributed to the judicious use and property of near-infrared (NIR) light emission of PLNPs and their preparation method, which together allow for a significant reduction in background noise. This technological advancement represents a significant step toward the development of more accurate and reliable glucose sensors, opening new avenues for non-invasive glucose monitoring in clinical settings and biomedical research.

## 4. Conclusion

The present study, based on the covalent grafting of GOx onto ZGO NPs, shows a tenfold lower LOD than that of our previous work using non-functionalized ZGOs. This improved sensitivity is likely to be due to a more efficient production of H₂O₂ near the ZGO surface, as well as an optimization of the ZGO-GOx preparation and assay protocol, resulting in a significant amplification of the signal. The achievement of such a low detection limit can also be attributed to the NIR light emission of the PLNPs, which allows a significant reduction in background noise. By optimizing the preparation of the NPs, we were able to identify ZGO2

(synthesized at 120 °C for 12 h), which after covalent functionalization with GOx, were able to detect glucose with a LOD of 0.01 µM in 1% human serum, after only 30 min of incubation.

The efficacy of the present technique lies in its high selectivity, ease of use, cost-effectiveness, and exceptional sensitivity, making it particularly suitable for regular monitoring of glucose levels in human serum, providing a practical and economical solution for glycemic assessment and control in clinical settings and biomedical research. In addition, the versatility of our detection system paves the way for adapting this technology to diagnose a wide range of other biomolecules associated with H₂O₂ production, including triglycerides, uric acid, and cholesterol. This adaptability greatly expands the potential applications of this method in clinical analysis and medical diagnosis.

## Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

## Acknowledgements

This research was funded by Agence Nationale de la Recherche (ANR-22-CE09-0029-01 PLEaSe). The figure of the protocol was made with the free version of Biorender.

## Conflict of Interest

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## Keywords

biosensors, functionalization, glucose, glucose oxidase, in vitro, PLNPs, ZGOs

Received: September 2, 2024

Revised: November 27, 2024

Published online: January 17, 2025

[1] Y. Wang, B. Li, T. Tian, Y. Liu, J. Zhang, K. Qian, *TrAC, Trends Anal. Chem.* **2022**, *149*, 116565.

[2] N. Gao, W. Zhou, X. Jiang, G. Hong, T.-M. Fu, C. M. Lieber, *Nano Lett.* **2015**, *15*, 2143.

[3] I. Ahmed, N. Jiang, X. Shao, M. Elsherif, F. Alam, A. Salih, H. Butt, A. K. Yetisen, *Sens. Diagn.* **2022**, *1*, 1098.

[4] C. Srichan, W. Srichan, P. Danvirutai, C. Ritsongmuang, A. Sharma, S. Anutrakulchai, *Sci. Rep.* **2022**, *12*, 1769.

[5] A. S. Bolla, R. Priefer, *Diabetes Metab. Syndr.* **2020**, *14*, 739.

[6] C. Gordon, *Br. J. Nurs.* **2019**, *28*, 434.

[7] N. Ahmadian, A. Manickavasagan, A. Ali, *J. Med. Eng. Technol.* **2023**, *47*, 121.
[8] M. Caldara, J. Kulpa, J. W. Lowdon, T. J. Cleij, H. Diliën, K. Eersels, B. V. Grinsven, *Chemosensors* **2023**, *11*, 32.

[9] S. R. S. Pour, D. Calabria, A. Emamiamin, E. Lazzarini, A. Pace, M. Guardigli, M. Zangheri, M. Mirasoli, *Chemosensors* **2023**, *11*, 546.

[10] M. Majdinasab, in *Wearable Biosensing in Medicine and Healthcare* (Ed: K. Mitsubayashi), Springer Nature Singapore, Singapore **2024**, pp. 35–66.

[11] A. Ko, C. Liao, *Anal. Methods* **2023**, *15*, 4377.

[12] T. Kant, K. Shrivas, A. Tejwani, K. Vandey, A. Sharma, S. Gupta, *Nanoscale* **2023**, *15*, 19016.

[13] H. W. Harshita, S. K. Kailasa, *Luminescence* **2023**, *38*, 954.

[14] H. N. Pham, M. H. Luong, D. T. Nguyen, Q. K. Nguyen, Q. A. Hoang, C. D. Sai, T. D. Thuy Ung, B. Pham, T. A. Huong Nguyen, D. T. Pham, T. N. Mai Pham, *Mater. Chem. Phys.* **2024**, *311*, 128562.

[15] M. Govindaraj, A. Srivastava, M. K. Muthukumaran, P.-C. Tsai, Y.-C. Lin, B. K. Raja, J. Rajendran, V. K. Ponnusamy, J. Arockia Selvi, *Int. J. Biol. Macromol.* **2023**, *253*, 126680.

[16] D. Yang, C. Cai, K. Liu, Z. Peng, C. Yan, J. Xi, F. Xie, X. Li, *J. Mater. Chem. B* **2023**, *11*, 7582.

[17] L. Liang, J. Chen, K. Shao, X. Qin, Z. Pan, X. Liu, *Nat. Mater.* **2023**, *22*, 289.

[18] L. Yang, S. Gai, H. Ding, D. Yang, L. Feng, P. Yang, *Adv. Opt. Mater.* **2023**, *11*, 2202382.

[19] J. Liu, T. Lécuyer, J. Seguin, N. Mignet, D. Scherman, B. Viana, C. Richard, *Adv. Drug Delivery Rev.* **2019**, *138*, 193.

[20] S. Wu, Y. Li, W. Ding, L. Xu, Y. Ma, L. Zhang, *Nano-Micro Lett.* **2020**, *12*, 70.

[21] K. Huang, N. Le, J. S. Wang, L. Huang, L. Zeng, W. Xu, Z. Li, Y. Li, G. Han, *Adv. Mater.* **2022**, *34*, 2107962.

[22] X. Zhao, X.-M. Gao, T.-Y. Gu, K.-L. Chen, Z.-Y. Yan, L.-J. Chen, X.-P. Yan, *TrAC, Trends Anal. Chem.* **2023**, *168*, 117279.

[23] T. Zhao, R. Abdurahman, R. Aiwaili, S. Wu, X.-B. Yin, *Coord. Chem. Rev.* **2023**, *488*, 215171.

[24] S.-K. Sun, H.-F. Wang, X.-P. Yan, *Acc. Chem. Res.* **2018**, *51*, 1131.

[25] N. Liu, X. Chen, X. Sun, X. Sun, J. Shi, *J. Nanobiotechnol.* **2021**, *19*, 113.

[26] U. Mushtaq, I. Ayoub, V. Kumar, V. Sharma, H. C. Swart, E. Chamanehpour, H.-G. Rubahn, Y. K. Mishra, *Mater. Today Bio* **2023**, *23*, 100860.

[27] J. Li, X. Huang, X. Zhao, L. Chen, X. Yan, *Angew. Chem., Int. Ed.* **2021**, *60*, 2398.

[28] J. Liu, B. Viana, N. Mignet, D. Scherman, Y. Liu, C. Richard, *Small* **2023**, *19*, 2303509.

[29] L. Wang, J. Shi, P. Wang, R. Rong, *Chem. Eng. J.* **2024**, *486*, 150291.

[30] Z. Ferjaoui, J. Liu, D. Scherman, N. Mignet, C. Richard, *Chem-NanoMat* **2024**, *10*, 202400078.

[31] T. Lécuyer, M.-A. Durand, J. Volatron, M. Desmau, R. Lai-Kuen, Y. Corvis, J. Seguin, G. Wang, D. Alloyeau, D. Scherman, N. Mignet, F. Gazeau, C. Richard, *Nanoscale* **2020**, *12*, 1967.

[32] Y. Jiang, Y. Li, C. Richard, D. Scherman, Y. Liu, *J. Mater. Chem. B* **2019**, *7*, 3796.

[33] T. Maldiney, A. Bessière, J. Seguin, E. Teston, S. K. Sharma, B. Viana, A. J. J. Bos, P. Dorenbos, M. Bessodes, D. Gourier, D. Scherman, C. Richard, *Nat. Mater.* **2014**, *13*, 418.

[34] T. Maldiney, M. U. Kaikkonen, J. Seguin, Q. Le Masne De Chermont, M. Bessodes, K. J. Airenne, S. Ylä-Herttuala, D. Scherman, C. Richard, *Bioconjugate Chem.* **2012**, *23*, 472.

[35] N. S. Rashed, S. Zayed, A. Abdelazeem, F. Fouad, *Microchem. J.* **2020**, *157*, 105069.

[36] G. Tiwari, R. Tiwari, *Pharm. Methods* **2010**, *1*, 25.

[37] H. Bao, Y. Pan, Y. Ping, N. G. Sahoo, T. Wu, L. Li, J. Li, L. H. Gan, *Small* **2011**, *7*, 1569.

[38] Y. E. Serge-Correales, D. Neumeyer, S. Ullah, R. Mauricot, Q. Zou, S. J. L. Ribeiro, M. Verelst, *Langmuir* **2023**, *39*, 1495.

[39] A. Lassenberger, A. Scheberl, A. Stadlbauer, A. Stiglbauer, T. Helbich, E. Reimhult, ACS Appl. Mater. Interfaces **2017**, *9*, 3343.

[40] S. M. Neacşu, M. Mititelu, E. A. Ozon, A. M. Musuc, I. D. M. Iuga, B. N. Manolescu, S. Petrescu, J. P. Cusu, A. Rusu, V.-A. Surdu, E. Oprea, D. Lupuliasa, I. A. Popescu, *Pharmaceuticals* **2024**, *17*, 690.

[41] M. A., A. Rajan S. N. K. Sahu, *Mater. Res. Express* **2020**, *7*, 094001.

[42] Y. Hui, L. Nan, X. Jing-Zhong, Z. Jun-Jie, *Chin. J. Chem.* **2005**, *23*, 275.

[43] B. B. Srivastava, A. Kuang, Y. Mao, *Chem. Commun.* **2015**, *51*, 7372.

[44] M. M. Can, G. Hassnain Jaffari, S. Aksoy, S. I. Shah, T. Fırat, *J. Alloys Compd.* **2013**, *549*, 303.

[45] F. Chen, G. Wang, J. I. Griffin, B. Brenneman, N. K. Banda, V. M. Holers, D. S. Backos, L. Wu, S. M. Moghimi, D. Simberg, *Nat. Nanotechnol.* **2017**, *12*, 387.

[46] C. Ji, J. Tan, Q. Yuan, *Chin. J. Chem.* **2021**, *39*, 3188.

[47] K. A. Altammar, *Front. Microbiol.* **2023**, *14*, 1155622.

[48] M. Mahmoudi, M. P. Landry, A. Moore, R. Coreas, *Nat. Rev. Mater.* **2023**, *8*, 422.

[49] V. P. Shah, K. K. Midha, J. W. A. Findlay, H. M. Hill, J. D. Hulse, I. J. McGilveray, G. McKay, K. J. Miller, R. N. Patnaik, M. L. Powell, A. Tonelli, C. T. Viswanathan, A. Yacobi, *Pharm. Res.* **2000**, *17*, 1551.

[50] X. Chen, Z. Yang, Q. Chen, Y. Zhang, *Front. Bioeng. Biotechnol.* **2023**, *11*, 1168086.

[51] Y. Tang, Q. Yang, T. Wu, L. Liu, Y. Ding, B. Yu, *Langmuir* **2014**, *30*, 6324.

[52] P. Wu, Y. He, H.-F. Wang, X.-P. Yan, *Anal. Chem.* **2010**, *82*, 1427.

[53] Y. Liu, D. Tu, W. Zheng, L. Lu, W. You, S. Zhou, P. Huang, R. Li, X. Chen, *Nano Res.* **2018**, *11*, 3164.

[54] S. Aliakbarpour, M. Amjadi, T. Hallaj, *Food Chem.* **2024**, *432*, 137273.

[55] S. Chatterjee, K. Kamalasekaran, S. Malaiappan, A. K. Sundramoorthy, *Inorg. Chem. Commun.* **2024**, *161*, 112140.

[56] X. Wang, C. Jia, S. Wang, Y. Dong, *Talanta* **2024**, *270*, 125584.