Micro‐Scaling Metal‐Organic Framework Films through Direct Laser Writing for Chemical Sensing

A zeolitic imidazolate framework (ZIF‐8) containing two enzymes that form a cascade biocatalyst has been integrated with 3D structures that are fabricated through a two‐photon polymerization using direct laser writing lithography. Glucose oxidase (GOx) and horseradish peroxidase (HRP) are encapsulated by the biomimetic self‐assembly process of ZIF‐8 to form the GOx/HRP/ZIF‐8 composite which grows in situ on the surface of microprinted carbon‐coated hexagonal substrates (cHS). The GOx/HRP/ZIF‐8/cHS film is applied to glucose detection through enzymatic oxidation of glucose to gluconic acid, forming H2O2, in the presence of GOx and the subsequent reduction of H2O2 to water in the presence of HRP and Amplex Red. This reaction is monitored using fluorimetry as the oxidation of Amplex Red with H2O2 catalyzed by HRP forms red‐emitting resorufin. The GOx/HRP/ZIF‐8/cHS film responds to glucose concentrations with a linear range of 10–200 µm, which correlates to the salivary glucose level in healthy humans. The GOx/HRP/ZIF‐8/cHS film shows more than eight times and 13 times enhanced activity than GOx/HRP/cHS (without ZIF‐8) and GOx/HRP/ZIF‐8 on a non‐3D patterned substrate, respectively. The GOx/HRP/ZIF‐8/cHS film retains more than 80% or 100% of its initial activity after being stored for over 2 months at ≤24 or 4 °C, respectively.


Introduction
Direct laser writing (DLW) lithography is an advanced 3D printing technology that uses femtosecond laser pulses and a multiphoton absorption process to rapidly fabricate intricate 3D structures with nanoscale resolution. [1]Compared to traditional lithography techniques such as X-ray, electron beam, and ultraviolet lithography, DLW lithography offers advantages in terms of speed, maintenance, fabrication efficiency, and control over the dimensionality of microfabricated structures. [2]5][6] Metal-organic frameworks (MOFs) are a renowned class of intelligently designed organic-inorganic hybrid materials with high crystallinity, organized pore structures, large internal surface areas, and pore volumes.9][10] MOFs can immobilize multiple enzymes and facilitate cooperative reactions between them by bringing their active sites close together.[13][14][15] The incorporation of a MOF biocatalytic cascade sensing platform with miniaturized 3D structures that are microfabricated through the DLW technique could accelerate the development of portable sensors that can promote personal health monitoring.
Here, a method for the simple fabrication of a MOF sensing platform supported on micrometer-sized structures has been developed by combining the facile synthesis of polycrystalline MOFs and porous composites with the flexibility of controlling dimensionality using the microfabrication capability of DLW lithography.For the first time, a two-enzyme biocatalytic cascade MOF composite is integrated with 3D microfabricated structures that were fabricated using DLW lithography.The zeolitic imidazolate framework (ZIF-8) is a well-studied MOF that has been selected in this study as the porous host for the biocatalytic Scheme 1. Schematic showing the typical DLW fabrication steps for enzymes/ZIF-8 composite-loaded microfabricated structures.The near-infrared laser beam at 780 nm was tightly focused through a microscope objective into the photoresist to fabricate the microstructures and the film was then flipped over for post-fabrication processing.The surface of the microprinted HS pattern was coated with carbon in Step 1 to obtain the cHS film that was then submerged in the precursor solution of ZIF-8, glucose oxidase (GOx), and horseradish peroxidase (HRP) in Step 2 to obtain the GOx/HRP/ZIF-8/cHS film.This scheme also applies to the fabrication of other ZIF-8 composite-coated 3D structures.
cascade reaction and to demonstrate the convenient integration of MOFs with the microprinted structures due to the facile synthesis, biocompatibility, and ultrahigh porosity of ZIF-8. [7,16]he enzymes used in this study are glucose oxidase (GOx) and horseradish peroxidase (HRP).GOx, also known as -D-glucose: oxygen 1-oxidoreductase, is widely used in the fabrication of biosensors for monitoring glucose levels in body fluids. [17]Ox is a dimeric protein that catalyzes the oxidation of -Dglucose to gluconic acid using molecular oxygen as an electron acceptor with simultaneous hydrogen peroxide production. [18]RP is a heme-containing enzyme that catalyzes the oxidation of substrates by hydrogen peroxide and subsequently reduces hydrogen peroxide to water. [19]The Fe center of heme has five different oxidation states to drive the biocatalytic reactions. [20]The immobilization of both GOx and HRP within the ZIF-8 structure achieves the oxidation of glucose and of a non-fluorescent Amplex Red indicator, which is oxidized by hydrogen peroxide in the presence of the HRP biocatalyst to yield a highly fluorescent resorufin molecule that is detectable through fluorescence spectroscopy.The current study demonstrates the potential of an integrated DLW-fabricated module with the biocatalytic ZIF-8 composite to create a fluorescent sensing platform featuring micro-scale dimensions with remarkable sensitivity that enables the detection of glucose levels within the salivary glucose range.Such optical miniaturized platforms could fast-track the development of practical MOF-based prototype devices to promote personal diagnostics with the potential to outperform their electrochemical counterparts. [21,22]

Fabrication and Characterization of the ZIF-8 and Biocatalytic Cascade GOx/HRP/ZIF-8 Composite Immobilized on 3D Microfabricated Structures
The decoration of 3D microfabricated structures with ZIF-8 and the GOx/HRP/ZIF-8 composite is described in Scheme 1.Three different structures, namely: hexagonal slab (HS), sodalite, and woodpile structures (see Figure S1, Supporting Information), were fabricated on indium tin oxide (ITO)-coated glass slides using the DLW.A drop of IP-S photoresist consisting of pentaerythritol triacrylate was first pipetted on the surface of an ITO glass substrate.Next, the substrate was placed on a 3D piezoelectric scanning stage that was positioned above a microscope objective.Laser light was directed through the objective and immersed into the liquid resist, inducing polymerization through the simultaneous absorption of two photons with a wavelength of 780 nm.The two-photon process facilitates deep laser penetration into the bulk of the photoresist that has negligible linear absorption in the near-infrared region to enable polymerization from a small focal volume.Additionally, the quadratic dependence of the polymerization rate on the intensity of the light ensures 3D spatial resolution. [2]After the structures have been developed, a graphitic carbon thin film was deposited on the surfaces to enhance their stability for post-fabrication processing, enabling the ZIF-8 deposition on the cHS film (Scheme 1, Step 1).Note that the carbon-coating step was crucial to the post-fabrication MOF deposition processes; without it, the microprinted structures either completely detached from the ITO surface or collapsed into the solution.The carbon film deposition was performed using conventional sputter coating. [25]The deposition of the ZIF-8 and enzymes/ZIF-8 composite was achieved in a one-step process by dipping the cHS film into the solution containing the precursors.To self-assemble GOx/HRP/ZIF-8, the carbon-modified structure on ITO was dipped into a solution containing zinc nitrate, 2-methylimidazole linker (2MiM), GOx, and HRP as indicated in Step 2 of Scheme 1 and detailed in the Experimental Section.An identical approach was taken to decorate the other microprinted structures with ZIF-8 and the GOx/HRP/ZIF-8 composite.
Figure 1a,b shows the scanning electron microscope (SEM) images of ZIF-8/cHS film at different magnifications by growing ZIF-8 particles at the surface of cHS film.ZIF-8 particles can grow on the carbon-modified structures due to the compatibility of the surface hydrophobicity of carbon and ZIF-8. [26,27]The triacrylate carbonyl groups in the chemical structure of the pentaerythritol triacrylate unit that makes up the microfabricated structures provide anchoring sites for ZIF-8 crystallites. [28]he growth of the GOx/HRP/ZIF-8 particles on the surface of cHS film is also a one-step protocol that was performed by dipping the film into an aqueous reaction mixture containing Zn 2+ , 2MiM, GOx, and HRP.The ZIF-8 crystal is built around the enzyme molecules leading to their encapsulation as illustrated in Figure S2, Supporting Information.Figure 1c is the SEM image showing the surface morphology of the GOx/HRP/ZIF-8/cHS films that have been fabricated by growing GOx/HRP/ZIF-8 particles directly on the cHS film from the reaction of the enzymes and 2MiM and Zn 2+ .The schematic for the biomineralization shown in Figure S2, Supporting Information indicates that the enzymes provide the anchoring sites to facilitate the self-assembly of ZIF-8 particles.Note also that ZIF-8 and GOx/HRP/ZIF-8 can be deposited on the surface of cHS film using other protocols (see Note S1 and Figure S3, Supporting Information).Although the entire area of the ITO was coated with carbon film, ZIF-8, and GOx/HRP/ZIF-8 particles selectively grow on the microfabricated structures confirming that the triacrylate carbonyl groups of the resin promote ZIF-8 MOF formation.Excess ZIF-8 and GOx/HRP/ZIF-8 particles can be removed through repeated rinsing of the MOF-decorated films in water.Despite the growth of GOx/HRP/ZIF-8 particles between the hexagonal structures of the GOx/HRP/ZIF-8/cHS film, seen in the SEM image in Figure 1c, the hexagonal micro print pattern is clearly marked.
The decoration of other carbon-modified structures with ZIF-8 and GOx/HRP/ZIF-8 was achieved by following the protocol described in Scheme 1 for the fabrication of the ZIF-8/cHS and GOx/HRP/ZIF-8/cHS films.Figure 1d,e shows the SEM images of the ZIF-8-coated sodalite lattice that indicates the selective growth of ZIF-8 particles on the sodalite lattice frame.A woodpile structure was also fabricated (see Figure S1c, Supporting Information), carbon-coated, and GOx/HRP/ZIF-8-modified.The SEM image of the GOx/HRP/ZIF-8/woodpile film shown in Figure 1f indicates that the GOx/HRP/ZIF-8 particles are grown on the stacked rods of the woodpile structure.
The energy dispersive X-ray (EDX) mapping in Figure 2 shows the elemental distribution in the ZIF-8/sodalite lattice, GOx/HRP/ZIF-8/cHS, and GOx/HRP/ZIF-8/woodpile structures.The EDX mapping of the ZIF-8/sodalite lattice shown in Figure 2a indicates the presence of elemental Zn and N, expected to be present in the ZIF-8, and elemental C which is attributed to the presence of ZIF-8, carbon film, and 3D-printed template.The oxygen contribution is expected to be solely from the 3D microprinted polymer structure.Furthermore, the elemental mapping of GOx/HRP/ZIF-8/cHS and GOx/HRP/ZIF-8/woodpile structures shown in Figure 2b,c, respectively, indicate the presence of P and Fe in addition to the elemental composition of ZIF-8.The P and Fe are present due to the GOx and HRP, respectively, in the GOx/HRP/ZIF-8-modified structures.Elemental P is present in the phosphate group of flavin adenine dinucleotide which is a coenzyme that is associated with GOx [18] and elemental Fe is in the heme group of HRP. [19]he porosity of the GOx/HRP/ZIF-8/cHS film could not be measured directly due to the small sample size with an active area of 1.4 mm 2 .The Brunauer-Emmett-Teller surface area (S BET ) and pore volume (V pore ) of the GOx/HRP/ZIF-8 powder (collected from the solution in which the films were formed, see N 2 sorption isotherms in Figure S4, Supporting Information) are 381 m 2 g −1 and 0.16 mL g −1 , respectively, which are lower than the pristine ZIF-8 (S BET = 1418 m 2 g −1 and V pore = 0.57 mL g −1 ).A reduced surface area is expected due to the presence of the enzymes. [7]o further show the presence of the enzymes in the GOx/HRP/ZIF-8/cHS film, GOx and HRP were labeled with FITC and RhB, respectively, to fabricate the dye-labeled d-GOx/HRP/ZIF-8/cHS film.Note that both the labeled and the unlabeled GOx/HRP/ZIF-8/cHS films were fabricated using an identical protocol.The confinement of both enzymes in the d-GOx/HRP/ZIF-8/cHS film was confirmed by fluorescence confocal microscopy imaging of the film to obtain fluorescent images from FITC-labeled GOx and RhB-labeled HRP incorporated in the film.Figure 3a shows green-fluorescent (i) and redfluorescent (ii) images of the d-GOx/HRP/ZIF-8/cHS film obtained after laser excitation of the film at 488 and 561 nm, respectively.The green-and red-fluorescent images correspond to the presence of FITC-labeled GOx (green) and RhB-labeled HRP (red) encapsulated within the d-GOx/HRP/ZIF-8/cHS film.The bright-field image of the d-GOx/HRP/ZIF-8/cHS film and the merged image of the three microscopy images are shown in Figure 3a-iii,iv, respectively.It is noteworthy that although the IP-S photoresist used to fabricate the 3D structures in this study is autofluorescent, [29] the carbon modification of the pris- Fourier-transform infrared (FTIR) spectroscopy was deployed to characterize the chemical functional groups present at the surface of the GOx/HRP/ZIF-8/cHS film.In the FTIR spectra shown in Figure 3c, the vibrations due to ZIF-8 and enzymes are distinguished with regions shaded in blue and red, respectively, while the contribution from cHS is unshaded.The absorbance of bond vibrations from the cHS resin support is highly intense, in contrast to the absorbance of bond vibrations from ZIF-8 and the enzymes.The peak at 1727 cm −1 that is the most prominent for all samples is the ─C═O stretching vibration of the triacrylate group of IP-S.Other vibrations at 1635 cm −1 , 1507 cm −1 and 1407 cm −1 are due to different stretching and bending modes of ─CH═CH 2 . [30]Both GOx and HRP are made up of peptides with identical IR peaks (red-shaded regions) at 1660 cm −1 and 1547 cm −1 as observed in the spectrum of the GOx/HRP/ZIF-8/cHS film.The band at 1660 cm −1 is an amide I vibration that occurs mainly from the ─C═O stretch of peptide linkages and a small contribution from the out-of-phase C─N stretching and the inplane NH bend, while the vibration at 1547 cm −1 is due to the combination of the NH in-plane bend and CN stretching of peptide groups. [31]The marked blue regions of the spectra represent vibrations due to ZIF-8 present in both the GOx/HRP/ZIF-8/cHS and ZIF-8/cHS but not on the cHS support.The typical IR bands of the imidazole ring are observed at 1584 cm −1 which appears at 1575 cm −1 in ZIF-8/cHS and could be assigned to the C═N stretching of the imidazole ring. [32]The peak at 1456 cm −1 corresponds to the entire imidazole ring stretching. [33,34]he crystallinity of the films was studied using X-ray diffraction (XRD) characterization and further confirms the presence of ZIF-8.The XRD patterns of the GOx/HRP/ZIF-8/cHS and ZIF-8/cHS films, Figure 3d, show peaks that correspond in position with the patterns of GOx/HRP/ZIF-8 and pristine ZIF-8 powders and the simulated pattern of ZIF-8; [35] there are no peaks from the cHS film within the range 2: 5°-20°.The (011) peak is more intense and narrower in the XRD pattern of the GOx/HRP/ZIF-8/cHS film than in the pattern of ZIF-8/cHS film, indicating preferential growth of the crystals [36] in the GOx/HRP/ZIF-8/cHS film.The presence of the enzymes likely induced the crystallization of ZIF-8, leading to the formation of larger crystals. [7]

Application of the Biocatalytic Cascade GOx/HRP/ZIF-8/cHS Film for Fluorescent Glucose Sensing
A cascade of oxidation reactions is initiated in the presence of both GOx and HRP embedded within the ZIF-8 structure after a drop of glucose-AR solution is placed at the surface of the GOx/HRP/ZIF-8/cHS film, as described in Scheme 2.
Catalyzed by GOx, glucose oxidation forms both gluconic acid and H 2 O 2 .AR is then oxidized by H 2 O 2 , catalyzed by HRP to produce resorufin, a red fluorescent dye that emits at 583 nm.The presence of the GOx/HRP/ZIF-8 particles that are assembled on a small 1.4 mm 2 area of cHS substrate (see Figure S6, Supporting Information) results in an intense fluorescent signal after exposure to the glucose-AR solution due to increased local concentration of resorufin.The initial response of the GOx/HRP/ZIF-8/cHS film to the presence of glucose was fast, within 30 s, as determined by the changes in the fluorescence intensity of resorufin produced from the oxidation of AR during the two-enzyme cascade reaction.Figure 4a-d shows representative spectra of the resorufin that was formed over 30 min at various glucose concentrations.The maximum emission wavelength ( em ) occurs at 583 nm in agreement with the range of published  em values of resorufin in the literature. [37,38]The increase in the fluorescence intensity with time confirms AR oxidation to resorufin from the H 2 O 2 produced during glucose oxidation and the enzymatic catalysis by HRP within the GOx/HRP/ZIF-8/cHS film.The gradual increase in fluorescence intensity is due to an increase in resorufin concentration.No further changes in the fluorescence intensity of resorufin were observed after 30 min, which suggests the completion of the AR oxidation reaction.
A solution contact with the GOx/HRP/ZIF-8/cHS film.This result suggests that it takes about 10 min of stabilization time to obtain a linear response from the GOx/HRP/ZIF-8/cHS film.The limit of detection of the GOx/HRP/ZIF-8/cHS film is 4.4 μm which was calculated from the limit of blank based on the approach described by Armbruster and Pry [39] and detailed in Note S2 and Figure S7, Supporting Information.The performance of the GOx/HRP/ZIF-8/cHS film exhibits a favorable comparison to some recent enzyme-encapsulated glucose sensors listed in Table S1, Supporting Information.Moreover, the range of linear response (10-200 μm) obtained from the GOx/HRP/ZIF-8/cHS film is clinically relevant to the range of salivary glucose levels (8-212 μm) found in healthy humans. [40,41]Fasting glucose concentrations above this range indicate the presence of diabetes mellitus.The GOx/HRP/ZIF-8/cHS film could be applied in developing a non-invasive and painless glucose monitoring device for personal health diagnostics.
The performance of the GOx/HRP/ZIF-8/cHS film was compared to those of control films: ZIF-8/cHS, GOx/ZIF8/cHS, HRP/ZIF-8/cHS, GOx/HRP/cHS, and GOx/HRP/ZIF-8/cNP (NP = non-patterned) to demonstrate the need for the dual enzyme in the biocatalytic cascade reaction, the importance of ZIF-8 for enzyme encapsulation and the need for the micropatterning to achieve high sensing response.The controls were synthesized under identical conditions and similar protein loading as the two-enzyme GOx/HRP/ZIF-8/cHS film, except for the ZIF-8/cHS film synthesized in the absence of enzymes (as detailed in the Experimental Section).A drop (10 μL) of a mixture consisting of 100 μm glucose and 5 μm AR prepared in phosphate buffer solution was added to the surface of the enzyme/ZIF-8-modified cHS patterns and ZIF-8/cHS pattern and the fluorescence was monitored at 30 min.As revealed in Figure 6a, the interaction of glucose-AR solution with either ZIF-8/cHS and GOx/ZIF-8/cHS films did not result in the formation of resorufin (as expected, since there is no HRP present).In contrast, the HRP/ZIF-8/cHS film yielded a low resorufin fluorescence, which has only 5% of the intensity generated by the GOx/HRP/ZIF-8/cHS film from the same reaction.The fluorescent emission generated in the presence of the HRP/ZIF-8/cHS film is likely an artifact due to the photooxidation of AR upon exposure to the excitation light due to the generation of the superoxide radical intermediate, O 2 ., in the aqueous glucose solution that is converted into H 2 O 2 , a substrate for HRP. [38]The results from these controls confirm that the cooperation of both GOx and HRP is required for biocascade catalytic sensing.
Figure 6a also shows the emission spectra of the GOx/HRP/cHS and the GOx/HRP/ZIF-8/cNP films after exposure to the same glucose-AR solution for 30 min.The GOx/HRP/cHS film was prepared by dipping cHS film in a vial containing GOx/HRP mixture but without adding zinc nitrate and 2-methylimidazole.After exposure to the glucose-AR solution for 30 min, the emission intensity of the GOx/HRP/cHS film (without ZIF-8) is about 12% of the intensity of the GOx/HRP/ZIF-8/cHS film.The GOx/HRP/ZIF-8/cNP film (see the SEM image of the film in Figure S8, Supporting Information) was prepared from the assembly of the GOx/HRP/ZIF-8 composite on the surface of a carbon-coated non-patterned (cNP) IP-S film.As indicated in Figure 6a, the emission intensity of the GOx/HRP/ZIF-8/cNP film after exposure to the glucose-AR solution, is about 30% of the GOx/HRP/ZIF-8/cHS film.The higher activity of the GOx/HRP/ZIF-8/cHS and the GOx/HRP/ZIF-8/cNP films over the GOx/HRP/cHS film   is due to the encapsulation of larger quantities of enzyme molecules within the ZIF-8 structure in contrast to deposition of the enzymes onto the pristine cHS film.The hexagonal patterned substrate (cHS) has a higher surface area-to-film-area ratio than the non-patterned (cNP) film, which increased the loading of GOx/HRP/ZIF-8 on the cHS film (see the SEM images in Figure 1c and Figure S8b, Supporting Information).Hence, a higher response of the GOx/HRP/ZIF-8/cHS film to the glucose-AR solution is expected due to increased glucose and GOx/HRP/ZIF-8 particle interactions compared to the GOx/HRP/ZIF-8/cNP film that has less particles.Also, the hexagonal patterned cHS surface is a 2D photonic crystal that has the ability to enhance fluorescence. [42,43]With careful manipulation of the photonic structure, the fluorescent response of the GOx/HRP/ZIF-8/cHS film could be improved further over the non-patterned GOx/HRP/ZIF-8/cNP film.Systematic investigations on the effects of periodicity of patterned films that are fabricated through the DLW on fluorescence sensing will be performed in the future to establish their ability to tune sensor responses.
To demonstrate the protective capability of the ZIF-8 encapsulation to the enzymes, the sensing performance of the GOx/HRP/ZIF-8/cHS film was tested in the presence of a proteolytic enzyme, trypsin.The emission spectra shown in Figure 6b indicate a slight decrease in the emission intensity after 30 min of exposure to the trypsin-containing glucose assay.Alternatively, the control, GOx/HRP/cHS film, which was also tested in the presence of trypsin under identical conditions showed a significant reduction in its fluorescence after exposure to the proteolytic enzyme.These results demonstrate that ZIF-8 acts as a protective barrier when encapsulating the GOx and HRP enzymes in the GOx/HRP/ZIF-8/cHS film thereby avoiding digestion by trypsin, otherwise, the enzymes were digested in the case of the GOx/HRP/cHS film since there was no cascade reaction to generate fluorescence.
The enzyme stability in the GOx/HRP/ZIF-8/cHS film was demonstrated after storing the film at refrigeration conditions (4 °C) and at room temperature (24 °C) for up to 60 days.The activity of the GOx/HRP/ZIF-8/cHS film was compared to that of the control, GOx/HRP/cHS film, which was stored under identical conditions; see Figure 6c for room temperature storage.The relative activity is defined as the percentage ratio of intensity of resorufin generated from each film to the intensity of resorufin generated from the GOx/HRP/ZIF-8/cHS film (at 0 day).The fluorescent responses of the GOx/HRP/cHS film to 100 μm glucose-AR solution were normalized to the response of the GOx/HRP/ZIF-8/cHS film (prepared at day 0) that had also been exposed to the same glucose solution.The response of the freshly prepared GOx/HRP/cHS film (shown as the blue bar, Figure 6c) is 12% that of the GOx/HRP/ZIF-8/cHS film (black bar); this result is consistent with the role ZIF-8 plays in encapsulating and stabilizing GOx and HRP.After 60 days, the GOx/HRP/cHS film showed a significant loss of performance, from 12% to 0.2%.On the other hand, the GOx/HRP/ZIF-8/cHS film showed above 80% retention in activity.Both films retained their performances under refrigerated conditions (Figure 6d).The effectiveness of ZIF-8 for bioencapsulation is demonstrated by the ability of the GOx/HRP/ZIF-8/cHS film to show sustained activity under unfavorable storage temperature conditions.Considering the potential of the encapsulated enzymes in the GOx/HRP/ZIF-8/cHS films for sensor application, the film stability at room temperature would allow for storage and transportation without significant degradation of performance.

Conclusion
A two-enzyme biocatalytic MOF composite was integrated with 3D structures that were fabricated through a two-photon polymerization based on the DLW lithography.The encapsulated enzymes, GOx and HRP, catalyzed the oxidation of glucose within the porous structures of ZIF-8 at the surface of the 3D microprinted structures.The GOx/HRP/ZIF-8/cHS films fabricated in this study were assessed for glucose sensing through the conversion of glucose to gluconic acid and H 2 O 2 in the presence of GOx and successive oxidation of the non-fluorescent AR to a fluorescent resorufin dye by H 2 O 2 in the presence of HRP.The GOx/HRP/ZIF-8/cHS film demonstrates a linear response to low glucose concentrations in the range of 10-200 μm, which is relevant for detection of salivary glucose levels in healthy humans.Moreover, the GOx/HRP/ZIF-8/cHS film was stable under refrigerated storage conditions (4 °C) for 2 months and showed decent fluorescence after storage at room temperature (≤ 24 °C) for 2 months.Based on the response of the GOx/HRP/ZIF-8/cHS film to low glucose concentrations and the high film stability over a prolonged duration, the film shows promise for the development of a non-invasive and painless optical glucose monitoring device.Based on the rapid microfabrication steps of the DLW technique and the simplistic MOF deposition, miniaturized sensors can be mass-produced from an industry perspective.
Potassium phosphate buffer (100 mm, pH 7.4) was prepared by dissolving KH 2 PO 4 (0.6696 g) and K 2 HPO 4 (2.687 g) in 200 mL Milli-Q water in a 250 mL capacity Schott bottle.Solutions containing different glucose concentrations (0.01 to 50 mm) and Amplex Red (AR, 10 μm) were prepared with the phosphate buffer solution and stored at 4 °C in the refrigerator.
The container of the AR solution was wrapped with Al foil for protection from ambient light.Enzyme solutions, GOx and HRP, were prepared by dissolving 11 mg of lyophilized powder of each enzyme in separate vials containing 2 mL Milli-Q water and were subsequently stored at 4 °C in the refrigerator.
DLW of Structures: The DLW process was performed in a Nanoscribe Photonic Professional GT system using IP-S resist.The templates for the DLW fabrication were designed with SOLIDWORKS 2018 in the Standard Tessellation Language format and converted into the General Writing Language format using the DeScribe application developed by the Nanoscribe for the 3D lithography.≈100 μL of IP-S was dropped on the surface of a 25 mm × 25 mm ITO glass substrate that was subsequently placed on the piezoelectric 3D scanning stage.The microscope objective immerses into the IP-S drop to focus the laser into the liquid resist and initiate its photopolymerization.The fabricated HS, woodpile, sodalite structures, and rectangular non-patterned (NP) IP-S were developed in a separate bath of 1-methoxy-2-propanol acetate (SU-8 developer) and isopropanol, followed by drying with a gentle flow of N 2 gas.The films were carbon-coated through the sputter coating of graphite using a Polaron E6700 carbon sputter coater.
Aqueous Synthesis of the GOx/HRP/ZIF-8/cHS, GOx/HRP/cHS, and GOx/HRP/ZIF-8/cNP Films: GOx/HRP were incorporated into ZIF-8 based on the protocol described by Wu et al. [22] for the synthesis of GOx/HRP/ZIF-8.An ITO glass substrate (1 cm × 1 cm) having a carboncoated HS (cHS) structure was rinsed with Milli-Q water.Then a mixture of Zn(NO 3 ) 2 solution (310 mm, 1 mL), GOx solution (5.5 mg mL −1 , 400 μL), and HRP solution (5.5 mg mL −1 , 600 μL) was prepared in a 5 mL capacity Eppendorf tube.A drop of the mixture was added to the printed area of the substrate for 2 min.Then the remainder of the mixture from the Eppendorf tube was transferred into a 22 mL clear glass.The Zn(NO 3 ) 2 /enzymeloaded substrate was carefully placed in the clear glass in a vertical position for another 15 min.To initiate the reaction forming GOx/HRP/ZIF-8, 2-methylimidazole solution (1.25 m, 10 mL) was pipetted into the glass vial, and the contents of the vial were allowed to react for 30 min without stirring at room temperature.The film from the reaction mixture was rinsed with ultrapure water (pH 7.02) by squirting water from a wash bottle onto the film on the substrate to remove excess crystals deposited around the hexagonal structure and dried at room temperature in the fume hood.The GOx/HRP/ZIF-8/cHS film was stored in the refrigerator at 4 °C and in an enclosed vial at 24 °C for more than 2 months to assess its stability.The GOx/HRP/ZIF-8 composite synthesis was repeated on the surface of a carbon-coated non-patterned (cNP) IP-S film by following the protocol described for the GOx/HRP/ZIF-8/cHS film.
For the synthesis of the GOx/HRP/cHS film (without ZIF-8), the above protocol was repeated but without the addition of Zn(NO 3 ) 2 and 2methylimidazole solutions, which were replaced with Milli-Q water.That is, a cleaned cHS substrate was immersed vertically in a mixture of GOx solution (5.5 mg mL −1 , 400 μL), HRP solution (5.5 mg mL −1 , 600 μL), and Milli-Q water (1 mL) in a 22 mL clear glass vial for 15 min.The solution in the vial was further diluted with 10 mL water, and the contents of the vial remained for another 30 min without stirring at room temperature.
Synthesis of GOx/ZIF8/cHS and HRP/ZIF-8/cHS: GOx/ZIF-8 and HRP/ZIF-8 composites were deposited at the surface of the cHS film using the same protocol described for the synthesis of the GOx/HRP/ZIF-8/cHS film.To synthesize the GOx/ZIF8/cHS film, a water-rinsed cHS substrate was immersed vertically in a mixture of Zn(NO 3 ) 2 solution (310 mm, 1 mL) and GOx solution (5.5 mg mL −1 , 1 mL) in a 22 mL clear glass vial for 15 min.2-methylimidazole solution (1.25 m, 10 mL) was pipetted into the glass vial, and the contents of the vial were allowed to react for 30 min without stirring at room temperature.The synthesis of the HRP/ZIF-8/cHS film was performed following the same protocol, except that HRP solution (5.5 mg mL −1 , 1 mL) was added to the ZIF-8 precursor instead of GOx solution.The films from each of the reaction mixtures were rinsed with ultrapure water (pH 7.02) by squirting water from a wash bottle onto the films on the substrates to remove excess crystals deposited around the hexagonal structure and were dried at room temperature in the fume hood.

Synthesis of ZIF-8/cHS Films and ZIF-8/sodalite Films:
The growth of ZIF-8 on the surface of the cHS film was performed by following the approach for the fabrication of the GOx/HRP/ZIF-8/cHS but without adding the enzymes. 1 mL of Zn(NO 3 ) 2 solution (310 mm, 1 mL) was prepared from which a drop was added to the printed area of the cHS film.The rest of the solution was transferred into a 22 mL clear glass and the Zn(NO 3 ) 2 loaded substrate was carefully placed in the clear glass in a vertical position for another 15 min.Then, 2-methylimidazole solution (1.25 m, 10 mL) was pipetted into the glass vial, and the reaction proceeded for 30 min without stirring at room temperature.The film from the reaction mixture was rinsed with Milli-Q water to remove excess crystals deposited around the hexagonal structure on the substrate and was dried at room temperature in the fume hood.
The deposition of ZIF-8 on the surface of the sodalite structure was performed using methanol as the solvent based on the protocol reported by Liu et al. [23] The ZIF-8 precursor solutions were prepared by dissolving 0.200 g of 2-methylimidazole in 2 mL of CH 3 OH in a 4 mL glass vial and 0.200 g of Zn(NO 3 ) 2 .6H 2 O was dissolved in 2 mL CH 3 OH in a separate 4 mL capacity glass vial.Both solutions were transferred to a 22 mL clear glass vial that already contained the woodpile or sodalite structure on ITO (1 cm × 1 cm), and the reaction proceeded for 2 h at room temperature.Next, the film was removed and washed with methanol from a wash bottle to remove excess ZIF-8 particles around the structures and then dried in the fume hood.
Dye Labeling of the GOx/HRP/ZIF-8/cHS Film: Dye-labeled GOx and HRP were prepared based on the protocol described in the literature. [15,24]eparate HRP and GOx solutions were prepared in 4 mL brown clear injection glass vials by dissolving 11 mg of each in 2 mL potassium bicarbonate buffer (KHCO 3 , 100 mm, pH 9).The dye solutions were prepared by dissolving fluorescein isothiocyanate (FITC, 10 mg) and rhodamine B (RhB, 10 mg) in dimethyl sulfoxide (1 mL, 99.9% Fisher Scientific) in separate 1.5 mL Eppendorf tubes.50 μL of FITC was pipetted into the brown glass vial containing GOx, while 50 μL of RhB was added to the brown glass vial containing HRP.Both vials were capped and covered with aluminum foil and stored at 4 °C in a refrigerator for 15 h.Next, the solution containing the dye-enzyme complex was placed in a Pur-A-lyzer maxi dialysis tube (12-14 kDa, Sigma Aldrich).The tagged enzyme was dialyzed against Milli-Q water (250 mL in a Schott bottle) for 18 h at 4 °C in a refrigerator.The dialysis procedure was performed four times, and the water was replaced each time until the water appeared colorless and no fluorescence of the dyes was observed in the dialysate.FITC-GOx and RhB-HRP were extracted back into new Eppendorf tubes, wrapped with Al foil, and stored in the refrigerator.The dye-labeled film, d-GOx/HRP/ZIF-8/cHS, was prepared by following the procedure described for the aqueous synthesis of the GOx/HRP/ZIF-8/cHS film, except that FITC-GOx and RhB-HRP were used instead of unlabeled GOx and HRP, respectively.
Fluorescence Sensing: The glass substrate coated with the GOx/HRP/ZIF-8/cHS film was attached to a microscope glass slide (Universal microscope glass slides, 1 mm thickness) and mounted on the spectrofluorometer sample stage at an angle of 22.5°to the excitation light.The excitation slit was set to a 2 nm bandpass stop so that the incident light was focused on the cHS pattern, which has an area of 1.4 mm 2 .A drop of glucose assay solution (10 μL) was pipetted to the surface of the GOx/HRP/ZIF-8/cHS film and was excited at 525 nm.The glucose drop consists of a mixture of glucose at different concentrations with 5 μm AR prepared in a 100 mm phosphate buffer at pH 7.4.For glucose sensing in the presence of trypsin, a drop of glucose assay (10 μL) consisting of 100 μm glucose, 5 μm AR, and trypsin (5.5 mg mL −1 ) prepared in phosphate buffer solution was pipetted to the surface of the GOx/HRP/ZIF-8/cHS film and allowed to react for 30 min before excitation at 525 nm.
Characterization: SEM images of the film were taken on a Zeiss Merlin ultrahigh-resolution SEM operated at a voltage of 5 kV equipped with an Ultim Extreme energy-dispersive X-ray spectroscopy detector for elemental mapping.The films were coated with an 8 nm iridium layer before imaging.The fluorescent images of the d-GOx/HRP/ZIF-8/cHS film were captured using the Nikon A1R Ti-E Confocal Fluorescent Microscope.Laser excitation of the film was performed at 405, 488, and 561 nm to obtain emis-sions at 450, 525, and 595 nm, respectively.For XRD characterization, the films were mounted with double-sided Kapton tape on top of poly(methyl methacrylate) plates into oversized sample holders before data collection.A Bruker D8 Advance A25 X-ray Diffractometer operating under CuK radiation (40 kV, 40 mA) equipped with a Lynx Eye XE-T detector was employed to obtain the X-ray diffractograms.Fourier transform infrared (FTIR) spectra were obtained in reflection mode on a Thermo Scientific Nicolet iN10 MX Infrared Microscope, and data were analyzed using OMNIC Picta Software.
Statistical Analysis: Linear regression was used to evaluate the statistical coefficient of determination (R 2 ) in the response of the GOx/HRP/ZIF-8/cHS film at different reaction times.Descriptive statistics were used to derive the mean and standard error in the analysis of the relative activity of the GOx/HRP/ZIF-8/cHS and GOx/HRP/cHS films over different storage duration and temperatures.Three samples of each of the GOx/HRP/ZIF-8/cHS and GOx/HRP/cHS films at different storage conditions were analyzed to yield triplicate measurements from which the mean and standard error were determined.For the calculation of the limit of detection (detailed in Supporting Information), standard deviations of 10 replicate measurements from the blank and the responses at a low concentration were derived using descriptive statistics.

Figure 2 .
Figure 2. SEM image and EDX mapping of a) the ZIF-8/sodalite structure at high magnification with corresponding elemental mapping as labeled for Zn, C, N, and O, b) the GOx/HRP/ZIF-8/cHS and c) the GOx/HRP/ZIF-8/woodpile with elemental mapping as labeled for Zn, C, N, O, P, and Fe.
tine microprinted structures prior to the deposition of ZIF-8 and GOx/HRP/ZIF-8 composites eliminates the background fluorescence (see Figure S5, Supporting Information).The fluorescence emission spectrum of the d-GOx/HRP/ZIF-8/cHS film showing the emission peaks of the FITC-labeled GOx at 525 nm and RhBlabeled HRP at 570 nm, Figure 3b, further supports the fluorescence microscopy images.

Figure 3 .
Figure 3. a) Confocal microscopy images of the FITC-labeled GOx and RhB-labeled HRP encapsulated in the d-GOx/HRP/ZIF-8/cHS film: i) excitation at 488 nm monitoring the fluorescence of the FITC-labeled GOx at 525 nm; ii) excitation at 561 nm and monitoring the fluorescence of the RhB-labeled HRP at 595 nm; iii) bright-field microscopy image; and iv) a merged image of the green, red and bright-field microscopy images.b) Fluorescent emission spectrum of the d-GOx/HRP/ZIF-8/cHS film at the excitation wavelength of 488 nm.c) FTIR spectra and d) XRD patterns of the GOx/HRP/ZIF-8/cHS (red line), ZIF-8/cHS (blue line), and cHS (black line) films.XRD patterns are also shown for the GOx/HRP/ZIF-8 (pink) and ZIF-8 (light blue) powders and simulated ZIF-8 (grey).

2 .
Scheme 2. Schematic of enzymatic cascade glucose sensing by GOx/HRP/ZIF-8/cHS film.O 2 oxidizes glucose in the presence of GOx to produce gluconic acid and H 2 O 2 .H 2 O 2 further oxidizes non-fluorescent Amplex Red in the presence of HRP to produce fluorescent resorufin which signals the response of the GOx/HRP/ZIF-8/cHS film.

Figure 4 .
Figure 4. Emission spectra of resorufin produced from the oxidation of AR indicating the time-dependent response of the GOx/HRP/ZIF-8/cHS film to different concentrations of glucose: a) 10 μm, b) 100 μm, c) 1 mm, and d) 10 mm for 30 min.

Figure 5 .
Figure 5. a) Fluorescence intensity-time plot of sensing response of the GOx/HRP/ZIF-8/cHS film to various glucose concentrations obtained at an emission wavelength of 583 nm, b) graph of the rate of formation of resorufin against glucose concentration, c) linear regression graph of fluorescence intensity against glucose concentration for the range 10-200 μm glucose, and d) graph of fluorescence intensity against glucose concentration for the range of 10 μm-10 mm glucose.