An improved nanocomposite

Abstract

This disclosure provides an improved nanocomposite comprising carbon nanodots (C-dots) and an enzyme co-encapsulated in a metal-organic framework. This disclosure also provides a method for forming the nanocomposite.

Description

Technical Field

[0001] This invention relates to an improved nanocomposite, particularly a nanocomposite comprising carbon nanodots and an enzyme encapsulated in a metal-organic framework. Background Technology

[0002] Enzymes are commonly used in sensors due to their efficient and selective role as catalysts. However, their commercial applications are severely limited by their stability issues and functional loss outside of optimal conditions, hindering applications that typically require enzyme reuse. Therefore, while enzymes have long been used as key identification elements in single-use sensors such as the well-known glucose sensor, significant efforts have been made to transform single-use analyte sensors into more reusable and long-term usable solutions.

[0003] In this regard, enzymes have been encapsulated in metal-organic frameworks (MOFs) to improve enzyme stability. While the MOF structure provides protection for the enzyme, it also leads to reduced catalytic activity compared to unencapsulated enzymes because it hinders analyte access to the enzyme's catalytic site. Therefore, this reduced activity makes it unsuitable for applications that may require rapid or real-time enzyme responses, such as electrochemical sensing.

[0004] Therefore, a method is needed to improve enzyme stability without affecting enzyme activity. Summary of the Invention

[0005] The present invention aims to address these problems and / or provide an improved nanocomposite, particularly for electrochemical sensing applications.

[0006] According to a first aspect, a nanocomposite is provided, comprising carbon nanodots (C-dots) and an enzyme co-encapsulated in a metal-organic framework (MOF).

[0007] The aforementioned C-dot can be any suitable C-dot. According to one specific aspect, the C-dot can be formed from an amino acid precursor. This amino acid precursor can be a positively charged amino acid precursor. For example, the amino acid precursor can be, but is not limited to, arginine, lysine, or a combination thereof.

[0008] The aforementioned nanocomposites can have suitable sizes. For example, nanocomposites can include an average diameter of 10-100 nm. Specifically, the average diameter of the nanocomposites can be 20-50 nm.

[0009] The aforementioned nanocomposite can be porous. For example, the nanocomposite can include an average pore size of 10-30 nm. Specifically, the average pore size can be approximately 20 nm.

[0010] The aforementioned nanocomposite can be included in an electrochemical sensor. According to one specific aspect, the nanocomposite can be included as a sensing layer in the electrochemical sensor.

[0011] The aforementioned electrochemical sensor can be any suitable electrochemical sensor. For example, an electrochemical sensor can be an electrochemical biosensor, more specifically a contact-type electrochemical biosensor.

[0012] The aforementioned electrochemical sensor can have a suitable duration of sustained sensitivity. Specifically, the electrochemical sensor can maintain ≥90% electrochemical sensitivity for at least one week. More specifically, the electrochemical sensor can maintain 100% electrochemical sensitivity for one week.

[0013] According to a second aspect, a method for forming a nanocomposite according to the first aspect is provided, the method comprising: Add carbon nanodots to a solution containing organic linkers; The enzyme was mixed with a solution comprising organic linkers and carbon nanodots to form a mixture; and Metal ions were added to the above mixture to form a nanocomposite.

[0014] The solution containing the organic linker described above can be any suitable solution. For example, the solution can be an aqueous solution.

[0015] The carbon nanodots added to the above solution can have a suitable size. Specifically, each added carbon nanodot can have an average diameter of ≤30 nm.

[0016] The added enzyme can be any suitable enzyme. For example, the aforementioned enzymes can include, but are not limited to, oxidoreductases, hydrolases, or mixtures thereof.

[0017] The aforementioned metal ion can be any suitable metal ion. According to one particular aspect, the metal ion can be, but is not limited to, transition metals, lanthanides, or combinations thereof. Attached Figure Description

[0018] To fully understand the invention and readily put it into practice, exemplary embodiments will now be described by way of non-limiting example only, with reference to the illustrative drawings. In the drawings: Figure 1 Thermogravimetric analysis (TGA) plots of various composites according to one embodiment are shown; Figure 2 Fourier transform infrared (FTIR) spectra of various complexes according to one embodiment are shown; Figure 3X-ray diffraction (XRD) patterns of various composites according to one embodiment are shown; Figure 4 The BET (Brunauer-Emmett-Teller) surface area of ​​various compounds according to one embodiment is shown; Figure 5 The pore sizes of various composites according to one embodiment are shown; Figure 6 A comparison of the encapsulation efficiency of various complexes according to one embodiment is shown; Figure 7 A comparison of the enzyme kinetics of various complexes according to one embodiment is shown; Figure 8 The average glucose sensitivity over one week on the rigid electrode is shown; Figure 9 The percentage change in glucose sensitivity over one week on the rigid electrode is shown; Figure 10 The average lactate sensitivity over one week is shown; Figure 11 The percentage change in lactate sensitivity over one week is shown; Figure 12 The average glucose sensitivity over one week is shown on the flexible printed electrode. Figure 13 The percentage change in glucose sensitivity over one week is shown on the flexible printed electrode; Figure 14 The enzyme activities of various complexes according to one embodiment are shown within a certain temperature range; Figure 15 The thermal stability of various composites over time according to one embodiment is shown; Figure 16 The stability of various complexes according to one embodiment in terms of residual enzyme activity after ethanol treatment is shown; Figure 17 The stability of various complexes according to one embodiment in terms of residual enzyme activity after trypsin treatment is shown; Figure 18 The percentage change in glucose sensitivity over 50 days of storage at 37°C is shown. Figure 19 The glucose sensitivity was shown after 50 days of storage at 37°C. Figure 20 A calibration diagram of a biosensor comprising a nanocomposite according to one embodiment is shown; Figure 21 The interference response of a nanocomposite according to one embodiment is shown; Figure 22 The percentage of glucose recovered from sweat is shown; Figure 23 A comparison of biosensor data with commercial assay results is shown; and Figure 24 The stability of glucose readings was demonstrated by reusing the electrode over a 30-day storage period. Detailed Implementation

[0019] As mentioned above, there is a need for an improved method to enhance enzyme stability without affecting enzyme activity.

[0020] In general, this invention provides an enzyme encapsulation within a metal-organic framework (MOF), which leads to improved enzyme stability. Specifically, the enzyme can be co-encapsulated with carbon nanodots (C-dots) into the MOF, thereby forming a nanocomposite comprising enzyme-carbon nanodots@MOF. The nanocomposite can be highly stable and exhibit enhanced electrochemical sensitivity. The enhanced sensitivity of the aforementioned nanocomposite is due to the peroxidase-mimicking activity of C-dots.

[0021] According to a first aspect, a nanocomposite is provided comprising carbon nanodots (C-dots) and an enzyme co-encapsulated in a metal-organic framework (MOF).

[0022] C-dots can be any suitable C-dot. Depending on a specific aspect, C-dots can be formed from amino acid precursors or nucleotide precursors. Amino acid precursors can be any suitable positively charged amino acid precursor. For example, amino acid precursors can be, but are not limited to, arginine, lysine, or combinations thereof. Specifically, C-dots can be formed from arginine. Even more specifically, C-dots can be arginine carbon dots (Argdots). Nucleotide precursors can be, but are not limited to, deoxyadenosine monophosphate.

[0023] The aforementioned nanocomposites can have suitable dimensions. For example, the nanocomposites can include an average diameter of 10-100 nm. For the purposes of this application, the average diameter refers to the average length of the longest dimension of the nanocomposite. Specifically, the average diameter of the nanocomposites can be 15-95 nm, 20-90 nm, 25-85 nm, 20-80 nm, 25-75 nm, 30-70 nm, 40-65 nm, 45-60 nm, or 50-55 nm. Even more specifically, the average diameter can be 20-60 nm, 28-53 nm, or more specifically, 28.1-52.6 nm. Compared to prior art nanocomposites that include enzymes encapsulated in MOFs, the average diameter of the aforementioned nanocomposites is reduced. For example, prior art nanocomposites can have an average diameter of 37.1-88.3 nm. When the nanocomposites are used in sensing applications, the reduction in the average diameter of the nanocomposites in the first aspect can thereby allow the analyte to diffuse into the enzyme more quickly.

[0024] The aforementioned nanocomposites can be porous. For example, the nanocomposites can include an average pore size of 10-30 nm. Specifically, the average pore size can be 15-25 nm, 18-22 nm, or 19-20 nm. Even more specifically, the average pore size can be approximately 20 nm.

[0025] Nanocomposites can have suitable shapes. For example, nanocomposites can have a defined shape. This shape can be, but is not limited to, spherical, hexagonal, or disc-shaped.

[0026] Nanocomposites can possess suitable zeta potentials. Specifically, the zeta potential of nanocomposites can be lower than that of a standalone MOF or C-dot encapsulated in a MOF without any enzyme. This demonstrates the co-encapsulation of enzymes and C-dot within the MOF structure.

[0027] The enzyme included in the nanocomposite can be any suitable enzyme. For example, the enzyme can be, but is not limited to, glucose oxidase (GO). x Lactate oxidase (LO) x ), cholesterol oxidase, uricase, urease, or a combination thereof. Specifically, the enzyme can be GO. x LO x Or a combination thereof.

[0028] The MOF included in the nanocomposite can be any suitable MOF. For example, the MOF can be, but is not limited to, a zeolite framework, including zeolite imidazolium ester frameworks (ZIFs), copper-based MOFs, Lavoisier Institute for Materials Research (MIL) based MOFs, or combinations thereof. Specifically, the MOF can be, but is not limited to, ZIF-8, ZIF-67, ZIF-60, Cu-MOF, MOF-88, Fe-MIL-100, or combinations thereof. Even more specifically, the MOF can be zeolite imidazolium ester framework-8 (ZIF-8).

[0029] Encapsulating enzymes with C-dot within MOFs enables the formation of composite materials with optimally sized nanocomposites, allowing for rapid and sensitive detection. Specifically, the MOF matrix can be modified, resulting in larger pore sizes and improved unimpeded diffusion of analytes into the enzyme. Furthermore, C-dot possesses inherent peroxidase mimicry capabilities, which can facilitate downstream catalysis of the generated catalase and electron transfer in subsequent amperometric detection. C-dot can also stabilize the enzyme's fragile 3D structure through non-covalent binding to prevent denaturation.

[0030] The aforementioned nanocomposite can be included in an electrochemical sensor. According to one specific aspect, the nanocomposite can be included as a sensing layer in the electrochemical sensor.

[0031] Electrochemical sensors can be any suitable electrochemical sensor. For example, an electrochemical sensor can be an electrochemical biosensor, more specifically a contact electrochemical biosensor. Electrochemical sensors can be used in a variety of sensing applications, such as, but not limited to, sweat testing, saliva testing, tear testing, urine testing, and interstitial fluid testing, to measure sweat glucose concentration, sweat lactate, uric acid, creatinine, and to name just a few.

[0032] The electrochemical sensor can maintain suitable sensitivity over an extended period of time. Specifically, the electrochemical sensor can maintain ≥90% electrochemical sensitivity for at least one week. More specifically, the electrochemical sensor can maintain 100% electrochemical sensitivity for one week.

[0033] The improved sensitivity of the nanocomposite can be attributed to the reduced average diameter, increased pore size (leading to reduced diffusion barriers), and / or the peroxidase-like activity contribution of the C-dot included in the nanocomposite compared to enzymes encapsulated in MOFs without C-dot. The reduced average diameter and increased pore size may be due to C-dot interfering with the biomolecule-induced rapid MOF formation, resulting in a reduced number of bonds per unit volume of sensing sample.

[0034] Electrochemical sensors, including those based on nanocomposites, can also exhibit improved stability over a certain temperature range. For example, electrochemical sensors can demonstrate stability and improved sensitivity within a temperature range of 40–80 °C.

[0035] The electrochemical sensor has now been generally described. It will be easier to understand the electrochemical sensor by referring to the following embodiments, which are provided in an illustrative manner and are not intended to be limiting.

[0036] Characterization of nanocomposites Various complexes are formed as follows: Argdot@ZIF-8 (i.e., a complex comprising C-dot formed from arginine precursor encapsulated in ZIF-8); GOx@ZIF-8 (i.e., a complex comprising enzyme GOx encapsulated in ZIF-8); and GOx-Argdot@ZIF-8 (i.e., a nanocomplex comprising enzyme GOx and C-dot formed from arginine precursor co-encapsulated in ZIF-8).

[0037] Thermogravimetric analysis (TGA) showed that GOx@ZIF-8 and GOx-Argdot@ZIF-8 exhibited almost identical weight loss patterns at 800°C, at 74.58% and 74.89%, respectively. Similarly, ZIF-8 and Argdot@ZIF-8 showed weight losses of 64.65% and 64.37% at 800°C, respectively, indicating that the complex could be stably formed even with the addition of Argdot. Figure 1 ).

[0038] The FTIR absorption peaks of GOx@ZIF-8 and GOx-Argdot@ZIF-8 largely correspond to the typical absorption peaks of ZIF-8. The imidazole -C=N- stretching occurs at 1590 cm⁻¹. -1 At this point, the imidazole ring stretching was determined to be at 1415 cm. -1 Location. At 1655 cm -1 The additional peak at that point indicates the amide I band of the protein, primarily from C=O stretching ( Figure 2 Therefore, the types of bonds present in the complex are largely the same, with the only significant difference being a decrease in the FTIR peak intensity of GOx-Argdot@ZIF-8. Similarly, XRD data show that the crystallinity of GOx-Argdot@ZIF-8 is essentially unchanged compared to the simulated ZIF-8. Figure 3 ).

[0039] To evaluate the surface area and porosity of the composite material, BET (Brunauer-Emmett-Teller) analysis showed that the specific surface area of ​​GOx@ZIF-8 was 238.71 m². 2Compared to / g, GOx-Argdot@ZIF-8 has a specific surface area of ​​198.19 m². 2 / g ( Figure 4 This is consistent with the increased pore size (19 nm) exhibited by GOx-Argdot@ZIF-8, which is approximately twice that of its counterpart GOx@ZIF-8. Figure 5 Therefore, the addition of carbon nanodots (C-dots) leads to an increase in pore size while maintaining a comparable specific surface area. This may be due to the widening of the channels through which analytes diffuse into the nanocomposite material, thus becoming a potential contributing factor to improved sensitivity.

[0040] Compared to enzymes encapsulated in pure MOFs, which are known to have high diffusion barriers, Argdot co-encapsulation lowers the diffusion barrier without increasing the complex size, thereby improving the overall electrochemical sensitivity.

[0041] Enzyme activity assays were then performed to ensure successful encapsulation of the enzymes into the MOF, showing encapsulation efficiencies of GOx@ZIF-8 and GOx-Argdot@ZIF-8 of 68.7% and 74.0%, respectively. Figure 6 Enzyme kinetic characterization showed that GOx-Argdot@ZIF-8 has a slightly smaller Kc than both free GOx and GOx@ZIF-8. M This indicates that the enzyme's affinity for the analyte is increased. Figure 7 ).

[0042] Electrochemical sensitivity of the complex Following composite characterization, extended sensitivity studies were conducted for electrochemical sensing applications. To investigate the effect of the GOx-Argdot@ZIF-8 nanocomposite on electrochemical sensitivity relative to typical ZIF-8 encapsulation, the nanocomposite was drop-fed onto a screen-printed commercial non-flexible carbon electrode to test the current response to increased sweat metabolite concentrations. The responses were measured and averaged over one week. The GOx-Argdot@ZIF-8 nanocomposite produced a significantly improved average response of 9.28 μA / mM / cm. 2 This is equivalent to its counterpart GOx@ZIF-8 ( Figure 8 Compared to the previous method, the detection range for glucose is improved by 40% to a maximum of 7 mM, which fully covers the typical range of glucose concentration in sweat.

[0043] The limits of detection (LODs) of GOx, GOx@ZIF-8, and GOx-Argdot@ZIF-8 were 21.7 μM, 35.9 μM, and 12.5 μM, respectively, indicating that GOx-Argdot@ZIF-8 possesses excellent sensitivity and a low LOD (LOD = 3σ / S). Furthermore, a one-week testing duration revealed its excellent stability (approximately 100% retained sensitivity), similar to that of GOx@ZIF-8, but in stark contrast to the exponential decrease in sensitivity of unencapsulated free Gox after the first round of testing. Figure 9 ).

[0044] The improved sensitivity stems from several factors, specifically the reduced nanocomposite diameter, the significantly increased pore size contributing to the reduction of the diffusion barrier, and the peroxidase-like activity of Argdot. The changes in composite diameter and pore size can be attributed to a decrease in the amount of bonds per unit volume of the sample, which may be caused by Argdot disrupting the rapid, biomolecularly induced formation of ZIF-8, as evidenced by the much lower peak intensity of the FTIR of the GOx-Argdot@ZIF-8 nanocomposite.

[0045] To further demonstrate that Argdot's enhancing ability is generally applicable to biosensors utilizing different enzymes, further optimizations were performed to synthesize LOx-Argdot@ZIF-8 nanocomposites using the same method as that used to form GOx-Argdot@ZIF-8 nanocomposites. These similarly demonstrated compatibility with LOx@ZIF-8 (10.2 μA / mM / cm²). 2 Compared to the LOx-Argdot@ZIF-8 electrode (14.8 μA / mM / cm), 2 The sensitivity increased by 1.4 times. Figure 10 ).

[0046] The detection range for lactate up to 1.80 mM was tested, while the LOD for all three samples was within 6–8 μM. Repeat tests showed that the LOx-Argdot@ZIF-8 nanocomposite maintained 100% sensitivity over one week, and free LOx decreased rapidly compared to LOx@ZIF-8, which showed a 10% decrease. Figure 11 ).

[0047] To test the application of the nanocomposite in wearable devices, additional tests were conducted on the use of the GOx-Argdot@ZIF-8 nanocomposite and its control on flexible printed electrodes to evaluate the transferability of the nanocomposite to different electrode carriers. The nanocomposite on the flexible electrode exhibited 8.49 μA / mM / cm². 2 Sensitivity ( Figure 12The sensor exhibited a 1.4-fold increase in sensitivity, with a detection range of 2.5 mM and a similar effect to previous commercial electrode tests. The same trend was also observed in sensor stability as in previous commercial electrode tests. Figure 13 Therefore, these data demonstrate the versatility and robustness of the GOx-Argdot@ZIF-8 nanocomposite for improving sensitivity in a variety of biosensing applications.

[0048] Stability of nanocomposites In addition to testing electrochemical sensing stability, an enzymatic assay was used to further evaluate thermal stability by exposing the nanocomposite and its control at 5°C intervals to temperatures ranging from 40°C to 80°C for 10 minutes. While free GOx lost 85% of its activity after incubation at 60°C, GOx@ZIF-8 and GOx-Argdot@ZIF-8 maintained full activity at incubation temperatures below 70°C. At 70°C, free GOx retained only 3.8% of its activity, while the encapsulated enzyme showed over 70% residual activity before degradation at 80°C. Overall, within the 10-minute incubation temperature range, the thermal stability trend of GOx-Argdot@ZIF-8 was comparable to that of GOx@ZIF-8, confirming that the increased sensitivity did not lead to compromised stability, unlike most existing MOF-based complexes. Figure 14 ).

[0049] The thermal stability test was extended to 6 hours at a fixed temperature of 55°C. The GOx-Argdot@ZIF-8 nanocomposite maintained 100% activity throughout the entire duration, compared to the control GOx@ZIF-8, which lost 18% activity after 3 hours and 34% activity after 6 hours. Meanwhile, free GOx showed the expected very drastic loss of activity, with 85% residual activity after 15 minutes and only 9.8% residual activity after 1 hour. Figure 15 ).

[0050] Therefore, compared to both the control GOx@ZIF-8 and free GOx, the GOx-Argdot@ZIF-8 nanocomposite exhibits superior thermal stability over time. While adding Argdot to the composite structure did not affect the thermal stability over 10 minutes within the experimental temperature range, the presence of Argdot significantly improved the thermal stability over time at 55 °C. Figure 15 Due to hydrolysis between the ZIF-8 ligand and ambient moisture, prolonged isothermal heating after partial decomposition of the ZIF-8 structure leads to denaturation of GOx in GOx@ZIF-8. In contrast, for GOX-ArgDot@ZIF-8, denaturation may be hindered by Argdot incorporation, which may be able to stabilize the 3D structure of GOx through non-covalent bonding.

[0051] Further stability tests were conducted on the samples and controls by exposing them to ethanol to evaluate the stability of the complex in organic solvents. GOx@ZIF-8 showed 70.5% residual activity, which is 1.9 times that of free GOx. Figure 16 The GOx-Argdot@ZIF-8 nanocomposite exhibited 78.4% residual activity, 8% higher than the residual activity of GOx@ZIF-8 and 2.1 times that of free GOx. Regarding exposure to trypsin treatment, both complexes retained 89.1% residual activity, 1.4 times that of free GOx. Figure 17 Therefore, both GOx@ZIF-8 and GOx-Argdot@ZIF-8 nanocomposites similarly improved the stability of the enzyme against organic solvents and trypsin degradation through protective encapsulation.

[0052] In addition to measuring enzyme activity, electrochemical assays further explored the retention of sensor sensitivity over 50 days after sample dropping onto the electrode and storage at approximately 37°C (body temperature). This assay was performed using repeated amperometric tests with the same set of electrodes. Both the control GOx@ZIF-8 and the GOx-Argdot@ZIF-8 nanocomposite retained full sensing capability for the first 32 days. On day 35, the GOx-Argdot@ZIF-8 nanocomposite retained 73.7% of its sensitivity, while GOx@ZIF-8 retained 66.7%. Subsequently, the retention sensitivity of both composites continued to show a steady decline, with the GOx-Argdot@ZIF-8 nanocomposite retaining an average of more than 6% of its initial sensitivity compared to GOx@ZIF-8. By day 50, the GOx-Argdot@ZIF-8 nanocomposite retained 36.8% of its initial sensitivity, while GOx@ZIF-8 retained 26.7%. In contrast, 35.7% of free GOx lost its activity by day 2, and 88.1% lost its activity by day 30. Figure 18 In short, this demonstrates that the GOx-Argdot@ZIF-8 nanocomposite retains 100% of its sensing capability for up to a month despite long-term storage and repeated measurements at body temperature. This stability is crucial for any long-term wearable or reusable sensor application.

[0053] More importantly, the daily sensitivity values ​​showed that the GOx-Argdot@ZIF-8 nanocomposite consistently produced an average sensitivity 1.4 times greater than the control throughout the entire 50-day trial. In the first 30 days, the average sensitivity of GOx@ZIF-8 was 6.82 μA / mM / cm. 2For a detection range up to 2.5 mM glucose, the GOx-Argdot@ZIF-8 nanocomposite exhibited an average sensitivity of 9.49 μA / mM / cm. 2 ( Figure 19 ).

[0054] Contact sensor for sweat testing The responses of each principal sample, including the free enzyme, the control complex GOx@ZIF-8, and the GOx-Argdot@ZIF-8 nanocomposite, were respectively measured using... Figure 20 Representative calibration curves are shown. As mentioned above, the GOx-Argdot@ZIF-8 nanocomposite exhibits a significantly improved electrochemical response compared to its control. Its linear range is 12.5 μM to 2500 μM (r0.05). 2 =0.998). Repeated calibration results confirmed the reproducibility of the sensor. The relative standard deviation (RSD) of five consecutive measurements within one week using the same electrode was calculated to be 3.4%, and the RSD of five different electrodes prepared and calibrated separately was calculated to be 4.9%. To confirm the selectivity and stability of the GOx-Argdot@ZIF-8 nanocomposite, interfering compounds commonly found in sweat, including lactate, uric acid, ascorbic acid, and potassium chloride, were added at relevant concentrations. Ampere testing showed that, before and after the addition of glucose, the GOx-Argdot@ZIF-8 nanocomposite-based biosensor selectively responded to glucose, while the response to all other interfering compounds was negligible ( Figure 21 ).

[0055] Sensor accuracy was also assessed by determining the current baseline and then adding a known concentration of glucose (1000 μM) dissolved in artificial sweat. Stable amperometric responses were recorded, and analyte concentrations were calculated using the previous calibration curve. When spiking was performed on the same day as calibration, the mean detectable concentration of the GOx-Argdot@ZIF-8 nanocomposite was 1016 ± 35 μM (repeated three times), equivalent to a recovery of 101.6%. On the same day, the recovery of free GOx was 94.7%, compared to 111.0% for the control GOx@ZIF-8. After repeating the spiking test exactly one week after calibration, the new current response was used in conjunction with the initial calibration to calculate analyte concentrations. In this case, the recovery of the GOx-Argdot@ZIF-8 nanocomposite remained sufficiently stable at 102.9%, while the recovery of GOx@ZIF-8 increased to 113.8%, and the recovery of GOx decreased to 55.4%, affecting the accuracy of the readings on day 7. Figure 22 The significant decrease in the percentage recovery rate of GOx within one week is also reflected in the above. Figure 22This reflects losses that could be even greater due to repeated testing throughout the week. Therefore, this data validates the improved stability, accuracy, and capability of the GOx-Argdot@ZIF-8 nanocomposite biosensor in detecting spiked glucose concentrations.

[0056] Following artificial sweat testing, real-world sweat glucose concentrations were measured using three pre-collected sweat samples from different individuals. Sweat concentrations were determined using a commercially available glucose assay kit prior to the amperometric test. Comparison of the GOx-Argdot@ZIF-8 nanocomposite biosensor readings with the measured values ​​revealed accuracies of 106.9%, 102.2%, and 104.4% for each sample, respectively. Figure 23 This confirms the accuracy and applicability of biosensors in actual sweat sensing.

[0057] To further evaluate the functionality of the GOx-Argdot@ZIF-8 nanocomposite, a contact fingertip sweat glucose assay was also designed for non-invasive resting-state measurements, even with low sweat volume. A simple porous PVA hydrogel was optimized to provide an interface and sweat collection capability, followed by a series of small clinical trials to obtain sensor data from three volunteers over 30 days. The GOx-Argdot@ZIF-8 nanocomposite electrode biosensors were fabricated as previously described, dried and stored at ambient temperature, and tested weekly for reuse over 30 days. For each test, each electrode had a fresh PVA hydrogel (1 cm diameter, 0.5 mm thickness) placed on it for sweat collection. Ampere readings were first recorded to obtain a baseline current. Simultaneously, sweat was allowed to accumulate on the volunteers' clean fingertips, an ideal testing location due to the high density of sweat glands (50–500 nL / cm²). -2 min -1 Sweat is released to obtain sufficient biofluid volume. After sweat accumulates, the fingertip comes into direct contact with the PVA hydrogel, causing the collected sweat to diffuse toward the enzyme. A second set of ampere readings is taken, and the increase in current amplitude compared to baseline is used to calibrate the data to determine glucose concentration.

[0058] Sensor data from three volunteers were collected over 30 days and averaged to determine the stability of the readings. Since all readings were consistent one hour after lunch over the month, it could be assumed that glucose concentration fluctuations were minimal in healthy individuals at the same time of day. The GOx-Argdot@ZIF-8 nanocomposite produced a highly consistent set of data, with the entire set of readings remaining between 94% and 104% of the average over 30 days. As for GOx@ZIF-8, readings showed a significant decrease, with the final reading being 76% of the original reading. As expected, by day 7, the second reading of free GOx showed an even more dramatic decrease of 55% (…). Figure 24 Therefore, the above experiments demonstrate the excellent ability of the GOx-Argdot@ZIF-8 nanocomposite as a highly stable enzyme nanocomposite to be repeatedly used in non-invasive biosensor applications over a long period of time.

[0059] According to the second aspect, a method for forming a nanocomposite according to the first aspect is provided, the method comprising: Add carbon nanodots to a solution containing organic linkers; The enzyme was mixed with a solution comprising organic linkers and carbon nanodots to form a mixture; and Metal ions were added to the above mixture to form a nanocomposite.

[0060] The solution including the organic linker can be any suitable solution. According to one particular aspect, the solution can be an aqueous solution. For example, the solution can be, but is not limited to, water, phosphate buffer, tris-buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer, or mixtures thereof.

[0061] The organic linker can be any suitable organic linker. For example, the organic linker group can be a conjugate base of a carboxylic acid or can include anions such as organophosphorus compounds, salts of sulfonic acids, heterocyclic compounds, and imidazoles. Specifically, the organic linker can include, but is not limited to, 2-methylimidazole, 4,4'-bipyridine, trimesic acid, terephthalic acid, or mixtures thereof.

[0062] The carbon nanodots added to the solution can have suitable sizes. According to one specific aspect, each added carbon nanodot can have an average diameter of ≤30 nm. Specifically, the carbon nanodots can have an average diameter of ≤10 nm. The carbon nanodots can have average diameters of 1-30 nm, 5-25 nm, 10-20 nm, and 12-15 nm.

[0063] Carbon nanodots can be any suitable carbon nanodot. For example, carbon nanodots can be as described above with respect to the first aspect.

[0064] The added enzymes can be any suitable enzyme. For example, the enzymes can include, but are not limited to, oxidoreductases, hydrolases, or mixtures thereof. Specifically, the enzymes can be, but are not limited to, oxidases, peroxidases, dehydrogenases, amide hydrolases, or mixtures thereof. Even more specifically, the enzymes can be, but are not limited to, glucose oxidase, lactate oxidase, cholesterol oxidase, uricase, sarcosine oxidase, lactate dehydrogenase, creatinine oxidase, and creatine kinase.

[0065] The aforementioned metal ion can be any suitable metal ion. According to one specific aspect, the metal ion can be, but is not limited to, transition metals, lanthanides, or combinations thereof. For example, the metal ion can be, but is not limited to, Zn. 2+ Cu 2+ Fe 3+ Zr 4+ Co 2+ Eu 3+ / Gd 3+ .

[0066] The addition of metal ions to a mixture can be carried out at any suitable temperature. For example, metal ions can be added to a mixture at a temperature of 4–37°C. Specifically, this temperature can be approximately 25°C.

[0067] Adding metal ions to the mixture is preferably accompanied by stirring for a predetermined period of time. Stirring can be carried out at a suitable speed. For example, the stirring can be carried out at 150-600 rpm. Specifically, stirring can be carried out at 200-550 rpm, 250-500 rpm, or 300-350 rpm. Even more specifically, stirring can be carried out at approximately 300 rpm. The predetermined period of time can be any suitable time period. For example, the predetermined period of time can be 10 minutes to 24 hours, 30 minutes to 12 hours, 1-10 hours, 2-8 hours, 3-5 hours, or 4-6 hours. Specifically, the predetermined period of time can be approximately 30 minutes.

[0068] This method can further include centrifuging the formed nanocomposite after adding metal ions to the mixture. Centrifugation after adding metal ions to the mixture can separate the formed nanocomposite from the reaction products. The centrifugation described above can include centrifuging the formed nanocomposite at a suitable speed. Specifically, centrifugation can be performed at speeds of 3000-10000 rpm, 4000-9000 rpm, 5000-8000 rpm, and 6000-7000 rpm. Even more specifically, centrifugation can be performed at a speed of 7500 rpm. Centrifugation can be performed at a suitable temperature. For example, the temperature can be 4-37°C. Specifically, the temperature can be approximately 25°C.

[0069] This method may further include washing the nanocomposite. Washing can be performed an appropriate number of times and with a suitable solvent. The solvent can be an aqueous solvent. For example, the solvent can be, but is not limited to, water, phosphate buffer solution, or Tris-buffer solution.

[0070] This method may further include depositing the nanocomposite onto the electrode surface to form a nanocomposite layer. Deposition can be performed by any suitable method. For example, deposition can be performed by, but is not limited to, drop casting, inkjet printing, ink dispensing, dip coating, or combinations thereof.

[0071] The electrodes described above can be any suitable electrodes. For example, the electrodes can be rigid, flexible, or stretchable electrodes.

[0072] The method may further include providing a protective layer on the nanocomposite layer. The protective layer may include one or more layers. Each of these layers may be the same as or different from each of the other layers included in the protective layer. The protective layer prevents the nanocomposite from leaching out of the nanocomposite layer over time and with repeated use of the electrodes.

[0073] According to one particular aspect, the protective layer may include a first protective layer comprising a combination of various polymers. These polymers may be, but are not limited to, chitosan, Nafion, or mixtures thereof. The protective layer may include a second protective layer disposed above the first protective layer and further away from the nanocomposite layer. The second protective layer may include a sweat-collecting gel. The sweat-collecting gel may include polyvinyl alcohol (PVA).

[0074] As can be seen, the method for forming the nanocomposite is a simple one. Furthermore, this method can be a one-pot process. Subsequent steps can be performed to form an electrode comprising the aforementioned nanocomposite.

[0075] The method has now been generally described, and it will be more easily understood by referring to the following embodiments, which are provided in an illustrative manner and are not intended to be limiting.

[0076] Synthesis of carbon nanodots 10 mL of amino acids (150 mM) dissolved in ultrapure water was added to a Teflon container and autoclaved at 180 °C for 12 hr. The carbon nanodots were then filtered using a 0.2 μm membrane filter. Prior to the synthesis of GOx-Argdot@ZIF-8, the carbon nanodots were further ultrafiltered using a 10 kDa MWCO centrifugal filter unit at 8000 rpm for 20 min. The amino acid precursors used included arginine, asparagine, and aspartic acid.

[0077] Synthesis of GOx@ZIF-8 and GOx-Argdot@ZIF-8 For the synthesis of GOx@ZIF-8, 2 mg of the enzyme GOx (or BSA control) was added to 1 mL of 2-methylimidazole (600 mM in water) and dissolved. Then, 1 mL of Zn(OAc)2 (40 mM in water) was added and vortexed. The solution was shaken at 300 rpm for 30 min at 23 °C to allow the complex to form, and then centrifuged at 3000 rpm for 10 min.

[0078] GOx@ZIF-8 pellets formed after centrifugation, allowing for the removal of the supernatant and subsequent washing with ultrapure water. After three washes, the pellets were resuspended in 700 μL of 50 mM Tris-HCl buffer at pH 8.8 in preparation for electrochemical assays.

[0079] The same procedure was used to synthesize GOx-Argdot@ZIF-8, with an additional 4 mg of Argdot dissolved in 2-methylimidazole before adding Zn(OAc)2. The encapsulated enzyme was similarly resuspended and stored at 4°C until dispensing. All of the above was applied to the synthesis of LOx@ZIF-8 and LOx-Argdot@ZIF-8, using 10 mg of LOx enzyme and the same concentration for all other reagents.

[0080] As described above regarding the first aspect, the formed nanocomposite was further characterized.

[0081] Sensor fabrication 3 μL of BSA@ZIF-8, GOx@ZIF-8, GOx-Argdot@ZIF-8, and free GOx were dropped onto the working electrode of a commercial C710 screen-printed electrode with a diameter of 4 mm. Next, 3 μL of SWCNT-chitosan (2 mg / mL SWCNT suspended in 1% chitosan dissolved in 2% acetic acid) was dropped onto the enzyme as a protective layer. An additional protective layer of 3 μL Nafion (1 wt% Nafion in water) was dropped onto the chitosan layer. A screen-printed flexible electrode (diameter = 3 mm) was prepared using the same protocol, with the additional step of dropping 3 μL of Prussian blue (30 mM) before sensor functionalization. The electrode was stored at 4 °C.

[0082] While exemplary embodiments have been described in the foregoing description, those skilled in the art will understand that many changes can be made without departing from the invention.

Claims

1. A nanocomposite comprising carbon nanodots (C-dots) and an enzyme co-encapsulated in a metal-organic framework (MOF).

2. The nanocomposite of claim 1, wherein, The C-dot is formed from an amino acid precursor.

3. The nanocomposite of claim 2, wherein, The C-dot is formed from a positively charged amino acid precursor.

4. The nanocomposite of claim 2 or 3, wherein, The C-dot is formed from arginine, lysine, or a combination thereof.

5. The nanocomposite of any one of the preceding claims, wherein, The nanocomposite has an average diameter of 10-100 nm.

6. The nanocomposite of any one of the preceding claims, wherein, The nanocomposite has an average pore size of 10-30 nm.

7. The nanocomposite according to any one of the preceding claims, wherein, The nanocomposite is included in an electrochemical sensor.

8. The nanocomposite according to claim 7, wherein, The electrochemical sensor is an electrochemical biosensor.

9. The nanocomposite according to claim 8, wherein, The electrochemical biosensor is a contact-type electrochemical biosensor.

10. The nanocomposite according to any one of claims 7 to 9, wherein, The electrochemical sensor has an electrochemical sensitivity of ≥90% for at least one week.

11. The nanocomposite according to claim 10, wherein, The electrochemical sensor maintains 100% electrochemical sensitivity for one week.

12. The nanocomposite according to any one of claims 7 to 11, wherein, The nanocomposite is included as a sensing layer in the electrochemical sensor.

13. A method for forming a nanocomposite according to any one of the preceding claims, the method comprising: Add carbon nanodots to a solution containing organic linkers; The enzyme is mixed with the solution comprising organic linkers and carbon nanodots to form a mixture; as well as Metal ions are added to the mixture to form the nanocomposite.

14. The method according to claim 13, wherein, The solution containing the organic linker is an aqueous solution.

15. The method according to claim 13 or 14, wherein, Each of the carbon nanodots has an average diameter of ≤30 nm.

16. The method according to any one of claims 13 to 15, wherein, The enzyme is an oxidoreductase, a hydrolase, or a mixture thereof.

17. The method according to any one of claims 13 to 16, wherein, The metal ions include transition metal ions, lanthanide ions, or mixtures thereof.

Images