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Gold Nanoparticle Encapsulated ZIF-8 for Mediator Free Enzymatic Glucose Sensor by Amperometry Anirban Paul, Gaurav Vyas, Parimal Paul, and Divesh N Narayan Srivastava ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00748 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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ACS Applied Nano Materials

Gold Nanoparticle Encapsulated ZIF-8 for Mediator Free Enzymatic Glucose Sensor by Amperometry Anirban Paul,a,b, § Gaurav Vyas,a,b,§ Parimal Paula,b,*, Divesh N. Srivastava a,b,* a

Analytical and Environmental Division and Centralized Instrument Facility,

b

Academy of

Scientific and Innovative Research (AcSIR), Council of Scientific & Industrial Research (CSIR), Central Salt and Marine Chemicals Research Institute (CSMCRI), G. B. Marg, Bhavnagar-364002, Gujarat, India. *Email- [email protected]. § Authors have contributed equally. Keywords: ZIF-8, gold nanoparticle, encapsulation, amperometry, enzymatic glucose sensor, tandem catalyst, Abstract A novel method has been developed for the encapsulation of gold nanoparticles and glucose oxidase together into the cavity of the ZIF-8 (Zeolitic imidazolate framework) in aqueous media and subsequently used for amperometric glucose sensing application. ZIF-8 is highly efficient, stable (thermally and chemically) microstructure and possess large surface area with a unique cavity to accommodate both the gold nanoparticles (AuNPs) and glucose oxidase (GOx). The as-synthesized composite is thoroughly characterized by various physicochemical methods and confirms the uniform distribution of the AuNPs within the metalorganic framework (MOF) cavity. The presence of highly conducting AuNPs enhance the activity of GOx and facilitate the mediator free tandem electrocatalytic reaction of both glucose oxidation and oxygen reduction. Further, the nano-composite exhibits admirable

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stability with low-level detection of glucose in aqueous media (50 nM of glucose) by using very low concentration of GOx (62 µg in 1 mL). 1. Introduction Diabetes mellitus is one of the most widespread chronic diseases occurs due to lack of physical activity, obesity and affects directly on the blood glucose levels which may lead to serious health complications like cardiac arrest, failure of kidney function and degeneration of neurons.1 This serious threat to health has increased in recent time significantly because of acquiring modern lifestyle and urbanization. Patients having this chronic disease are recommended to check their blood glucose level at a regular interval and prescribed insulin shot periodically for long-term management of their blood glucose level.2 However, the available methods are associated with inconvenience and pain as well as have possibilities of causing injections. Hence, non-invasive glucose monitoring is highly desirable.3–5 Ever since, Clark and Lyon first proposed the concept of glucose sensing in 1962, it is almost 55 years passed and still the search for new approaches are being welcomed by the scientific community. The development of amperometric glucose sensor devices can be divided into three generations. First-generation devices were based on the consumption of the atmospheric O2 co-substrate and the production and detection of H2O2. Second generation devices had been mainly focused on replacing atmospheric O2 with various electron mediator. Third generation devices had been designated with mediator free non-invasive and point of care detection of glucose from saliva or sweat. The whole journey has been extensively reviewed by various authors.6–8 Mediator free direct electron transfer between the active centre of redox protein and the electrode is one of the important and fundamental concepts to develop new biosensor and bio-electrocatalytic devices.9 Although, direct electron transfer to the naked/bare electrode is rather difficult to achieve as the FAD (Flavin adenine dinucleotide)/FADH2 and the prosthetic group of Glucose oxidase (GOx) are deeply


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embedded inside the non-conducting layer of rigid protein (GOX) structure. External electron mediator species is thus used to facilitate this catalytic reaction artificially.9,10 Recently, MOF has fascinated scientific fraternity by its unique design, the large surface area along with uniform, tuneable pore sizes11 and the thermal stability it can provide to the biomolecules. Among several zeolitic MOFs, ZIF-8 was reported to be an effective MOF, consists of specific pore size to accommodate biomolecule and protect them from major external stimuli.12–20 Metal nanoparticles had been used extensively for dual purpose as it significantly increases the surface area so that the electron tunnelling distance is sufficiently low and also to increase the surface conductance of the electrode to mediate charge easily to the electrode surface.21–26 Dong et al recently reported ZIF-8, modified with GOx and PVP stabilized NiPd nanostructure composite as an effective tandem catalyst for glucose sensing.27 This is the first report where it was claimed that both nanoparticles and glucose oxidase can be jointly encapsulated into ZIF-8 matrix, although there are few limitations of such microsystem which encouraged us to design this one-pot synthetic route in aqueous media. Since the semiconducting NiPd nanoparticle has been replaced by a metallic AuNPs, we could achieve very low detection of glucose (50 nm) compared to the LODs reported earlier for this type of system. Use of highly conducting AuNPs not only decreases the electron tunnelling distance but also increased the activity of the probe by overcoming the threshold barrier caused due to low conducting ZIF-8. Among several nanoparticles, citrate stabilized AuNPs had been proved to be a better choice over other nanoparticles. This is because of obvious reason of its high conductivity which helps to mediate charge from the electrolyte to electrode surface and moreover its highly dispersed nature in water causing zero interference with biological species. Hence, AuNPs was extensively used by the scientific community for amperometric detection of glucose.28–31



In this work, we report the synthesis of GOx@ZIF-8(AuNPs) by encapsulation of gold nanoparticles and glucose oxidase together into the cavity of ZIF-8 (scheme-1) and utilize for the catalytic oxidation of glucose. Citrate stabilized Au nanoparticle was synthesized and reported in our previous work

32

. Here it has been encapsulated successfully in to the ZIF-8

matrix along with glucose oxidase. The catalyst was utilized in amperometric glucose sensor having a detection limit as low as 50 nM of glucose, accrediting glucose monitoring from sweat. Further, the catalyst can not only oxidize the glucose, but also reduce the H2O2 by participating in oxygen reduction reaction. Oxidation of glucose was also confirmed by assaying gluconate with glucose oxidase-peroxidase standard colorimetric experiment. The switch over redox stability of the redox couple FAD/FADH2 makes the catalyst more novel towards sophisticated fabrication. In sort, the present catalytic protocol is more efficient and robust for the low-level detection of glucose (50 nM) in aqueous media based on our knowledge.

Scheme 1: Synthesis of GOx@ZIF-8(AuNPs) 2. Experimental: 2.1. Materials and methods All the chemicals were of analytical grade and used without further purification. HAuCl4.3H2O, trisodium citrate dehydrate, Glucose oxidase from aspergillus niger (GOx), 2-


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Methylimidazole were purchased from sigma Aldrich and used as received. Zn(NO3)2.6H2O was purchased for SRL chemicals and used as received. GOx@ZIF-8(AuNPs) nanocomposite was

synthesized at room temperature in aqueous media. To prepare GOx@ZIF-8(AuNPs) nanocomposite, AuNPs and GOx (aspergillus niger) were added in the reaction mixture of the ZIF-8Glassy carbon electrode of 5 mm diameter had been used for all electrochemical experiments. The electrode was polished with 0.3-micron alumina slurry followed by sonication in 1:1 ethanol-water mixture for 15 minutes. The electrode was then rinsed with ultrapure water and dried at room temperature. All the experiments were done in three electrode electrochemical setups comprising glassy carbon as working, Pt wire (0.1 mm diameter) as a counter and Ag/AgCl (sat. KCl) as a reference electrode. The GOx@ZIF8(AuNPs) slurry was prepared by dispersing 1 mg of material in 200 µL of pH 7.4 buffer and sonicated for 10 minutes. 5 µL 0.5% nafion is added to the mixture as a binder. The crystal structure of the product was characterized by XRD (PAN Analytical Empyrean Series 2 X-ray diffraction system). The morphology and microstructure of the synthesized materials were investigated by FESEM (JEOL JSM-7100F) and HRTEM (JEOL JEM-2100). The samples were dispersed in water followed by loaded over TEM grid (300 mess lacey carbon coated copper grid) and SEM mount (brass). UV-VIS spectra were recorded with Varian spectrophotometer. The BET surface area measurments were done on Micromeritics 3 Flex. Prior to the study the degasing was carriedout at LN2 temperature (-194°C) FT-IR was done at Parkin-Elmer G-FTIR. Detail synthetic procedure of GOx@ZIF-8(AuNPs) nanocomposite is as follows. For all electrochemical measurement Metrohm Autolab 204 potentiostat/galvanostat was used. 2.2. Synthesis of GOx@ZIF-8(AuNPs)



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ZIF-8 was synthesized by previous reported protocol.13 22.70 gm of 2methlyimidazole (108 mili moles) was taken in 400 mL of ultrapure mili-Q water. 50 mg of GOx (8170 units in 50 mg) was added to the same solution. This mixture was slightly yellow in colour due to dissolved GOx and labelled (A). 1.17 gm of Zn(NO3)2.6H2O (1.53 mili moles) was dissolved in another 390 mL of ultrapure milli-Q water separately and labelled (B). Gold nanoparticles (AuNPs) were prepared by our previously reported

protocol32.

Trisodium citrate dehydrate was used as stabilizing as well as a reducing agent in aqueous media. As synthesized AuNPs were stored at 4˚C temperature for further use. 10 mL of citrate stabilized AuNPs was taken separately (C). The mixture of B & C was added to the mixture A under constant stirring of 600 rpm at room temperature and the reaction mixture was continuously stirred for 3 hours. In the very beginning of the reaction, the solution was transparent and yellow in colour which started to turn hazy after few minutes of stirring. After completion of 3 hours, the mixture was allowed to stand for overnight and then the supernatant was decanted. It has been observed that the yellow colour of the mixture disappeared after the successive addition but no deep red colour of AuNPs appeared. This give initial confirmation on encapsulation of both GOx and AuNPs together inside ZIF-8 moiety. The settled solid part was centrifuged and washed with 50 mL of water for 3 times to remove any excess GOx and AuNPs (not encapsulated). Further it was dried under vacuum and the purple colour solid composite was recovered, preferably assumed the formation of GOx@ZIF-8(AuNPs) nanocomposite. The synthesized probe was characterized by FT-IR, Powder XRD, FE-SEM, STEM, and UV-Vis spectroscopy to confirm the artefact. 3. Results and Discussions 3.1 Characterization of Materials The citrate stabilized AuNPs and GOx material shows very distinctive features. Its phase was determined by X-Ray powder diffraction (P-XRD) after vacuum drying. The P-


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XRD pattern of as synthesized GOx@ZIF-8(AuNPs), is shown in Figure 1 (blue colour) and is compared with the simulated powder XRD data of the ZIF-8 (black colour), furnished in the same figure. It is clearly observed that the P-XRD peaks of the synthesized GOx@ZIF8(AuNPs) match well with the simulated ZIF-8, indicating no change in the native structural property of the ZIF-8 due to encapsulation of AuNPs and GOx. As synthesized GOx@ZIF8(AuNPs) has been confirmed by its cubic unit cell (JCPDS: 00-062-1030, 17.0116 Å, 90°). The scale was zoomed to visualize the presence of AuNPs and the standard peak was obtained (38.22○) correlating the presence of Au (111), depicted in the inset of Figure 1.

Figure 1. XRD patterns for simulated ZIF-8 (JCPDS: 00-062-1030) and as synthesized GOx@ZIF-8(AuNPs). Inset image: Standard peak of AuNPs designated attributing the presence of Au (111) plane (JCPDS: 00-066-0091).

Apart from comparing the JCPDS data the P-XRD data of only AuNPs and only GOx were also recorded and depicted in Figure S1 (See ESI) and Figure S2 (See ESI) respectively. Whereas the GOx can be seen as an amorphous moiety in recorded P-XRD and the AuNPs show several peaks. Among these peaks, the 100% peak appears at 38° 2θ. It is argued that only this 100% peak appears in P-XRD of the composite and other minor peaks are not visible ACS Paragon Plus Environment


due to low concentration of AuNPs present in the composite. It was observed that small peaks at ~21˚, 26˚ and 29˚ 2θ appeared in P-XRD, which is due to presence of unreacted 2-methyl imidazole trapped in the moiety.33 The morphology of GOx@ZIF-8(AuNPs) was examined by field-emission scanning electron microscopy (FESEM). Figure 2a represents assembled star shaped morphology of ZIF-8 microstructure at low magnification. Whereas high magnified FE-SEM image shows the assembly of rod like star morphology (Figure 2b). The general morphology of ZIF-8 is reported as polyhedral. The affinity of GOx towards imidazole containing building block (ZIF-8) arising from intermolecular H-bonding and hydrophobic interaction and this may be a driving force that the composite acquires rod oriented star shape morphology.27

2 nm

Figure 2. FESEM image of 5nm gold coated GOx@ZIF-8(AuNPs) (a) crystalline growth (b) single particle showing large surface area (c) High resolution HRTEM image of Au lattice fringes (d) STEM-EDX line profile of GOx@ZIF-8(AuNPs) showing almost equal distribution of AuNPs.


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LR-TEM was performed to observe distribution of AuNPs over ZIF-8 moiety. The result is depicted in Figure S3 (See ESI). Two different contrasts can clearly be seen in this figure. It is known that the low Z elements appears lighter in TEM whereas heavy metal appears darker due to amplitude contrast. Therefore it is argued that the lighter contrast corresponds to ZIF-8, whereas darker contrast to AuNPs. The HR-TEM was performed for detailed investigation of the synthesized microstructure and is depicted in Figure 2c. The image shows the presence of highly crystalline (111) plane of Au nanoparticles (lattice fringes) which also correlates with the XRD result. To confirm the encapsulation of Au nanoparticles within ZIF-8, STEM-EDX line profiling has been performed. The area selected for line profiling and corresponding line profile is depicted in Figure S4 (see ESI) and Figure 2d respectively, whereas its EDX spectra are provided in Figure S5 (see ESI). The result portrays homogeneous distribution of Au nanoparticle with an equivalent spatial distribution within large pore size of the ZIF-8 microstructure. BET N2 adsorptiondesorption isotherm study has been performed to check the porosity of the synthesized probe. The result is depicted in Figure S6. A large surface area of 113.6 m2/g had been observed for GOx@ZIF-8(AuNPs) which may validate the high surface coverage star shaped morphology obtained in FE-SEM. We also performed BET N2 adsorptiondesorption isotherm study of Au@ZIF-8 and a surface area of 4.7 m2/g had been observed. The result is depicted inset of Figure S6 (See ESI). Increase of surface area in the presence of GOx confirms the ability of ZIF-8 to possess tuneable pore size to accommodate large biomolecules. 34–37 To, prove the presence of GOx in its native structure inside the ZIF-8 cavity, Fourier Transform Infrared Spectroscopy (FT-IR) had been performed. GOx has characteristic IR peaks at 1654 and 1424 cm-1 due to stretching vibration of –C=O group present in the peptidic bond of amide-I and in-plane bending vibration of N5H in FAD group respectively.38 It is



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clearly seen from Figure 3 that 1654 cm-1 peak of amide-I is slightly shifted to 1644 cm-1 as it is highly sensitive towards the structural modification.39 Presence of both peaks at 1644 and 1424 cm-1 signify the presence of GOx. These results clearly suggest that the GOx does not degrade and is present in native structure.

Figure 3. FTIR Spectra of GOx@ZIF-8(AuNPs)possessing two distinguishable peaks at 1644 cm-1 for -C=O stretching of Amide I and 1424 cm-1 for plane bending of FAD group. 3.2 Assaying D-glucose with GOD-POD and HPLC: After thorough characterization, the activity of GOx inside the synthesized GOx@ZIF8(AuNPs) was investigated by conventional glucose oxidase-peroxidase (GOD-POD) assay test. In this test, Glucose oxidase (GOx) catalyses the oxidation of glucose to gluconate followed by generation of hydrogen peroxide (H2O2, equation 1 and 2), which is detected by a chromogenic oxygen acceptor, phenol, 4-Aminophenazone (4-AP) in the presence of peroxidase (POD)27:

!" #$


(1)

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+,

%ℎ# (4 )%) !" -. /0## .1

(2)

Characteristic absorbance peak at 505 nm wavelength confirms the presence and activity of GOx inside the probe. The outcome is depicted in Figure S7 (see ESI). No colour change has been observed in a control experiment where glucose was not added. We also examined interference effect with various other possible analytes like fructose, galactose, lactose and urea (see ESI Figure S8). For this purpose, we had mixed fix concentration of D-glucose in each of the analytes and measured the absorbance. The result is depicted in Figure S9 (see ESI). The result suggests that the synthesized GOx@ZIF-8(AuNPs) probe is very active because of zero interference in the biological environment. Moreover, reverse phase high performance liquid chromatography (RP-HPLC) was done to investigate the existence of gluconate generated on the catalytic reaction. For this purpose, 4 samples have been taken into consideration: GOx from Aspergillus Niger (A), Glucose (B), a mixture of GOx (Aspergillus Niger) and Glucose (C), a mixture of GOx@ZIF-8(AuNPs) and glucose (D). The result is depicted in Figure S10 (see ESI). Characteristic peaks for GOx (A) and glucose (B) can be seen in their respective spectra. In presence of GOx glucose is oxidized to gluconate and hence a new sharp peak had been observed at retention time 21.5 minutes (C). The synthetic probe (D) possesses an exactly common retention time with (C) at 21.5 minutes with ~90% similar peak intensity, depicts the occurrence of same catalytic reaction occurs for (C) . The result signifies that GOx, encapsulated into ZIF-8 does not degrade and possesses its native catalytic activity towards oxidation of glucose to gluconate in presence of natural oxygen. 3.3 Electrochemistry of GOx@ZIF-8(AuNPs) Cyclic voltammetry (CV) measurement was carried out at a scan rate of 100 mV/S to evaluate the electrochemical property of GOx@ZIF-8(AuNPs). For this purpose, three composites:

ZIF-8(AuNPs),

GOx@ZIF-8

and

GOx@ZIF-8(AuNPs)


were

scanned


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potentiodynamically. Prior to use, GCE was sonicated in 1:1 ethanol-water for 15 minutes and then properly polished in 10-micron alumina slurry and finally washed with milli-Q water and then dried. 5 mg of each composite was dispersed in 1 mL of 0.1 M pH 7.4 PBS buffer by ultrasonication for 10 minutes and 10 µL from the aliquot was drop casted onto the glassy carbon electrode (GCE) having a diameter of 5 mm. The electrode was kept covered to avoid external dust and was stored overnight at room temperature for drying. The modified electrode, projected as working electrode, was then immersed in N2 saturated 0.1 M pH 7.4 PBS buffer for CV measurement. Pt wire (0.1 mm diameter) was taken as a counter electrode, whereas Ag/AgCl (sat. KCl) was taken as a reference electrode. CV was measured in the range of -0.2 to -0.7V vs Ag/AgCl (sat. KCl) at a scan rate of 100 mV/S. Relatively higher scan rates are always preferred to assess the reversibility of electrochemical reaction. The result is depicted in Figure 4. The ZIF-8(AuNPs) exhibits typical non-faradaic capacitive nature due to double layer capacitance and hence indicative of the absence of any redox species (Figure 4, red dashed lines). CV of GOx@ZIF-8 possesses faradaic response due to the presence of FAD/FADH2 redox couple. Anodic peak (Epa) appeared at -395 mV whereas cathodic peak (Epc) appears at -502 mV. The peak separation (∆Ep) between cathodic and anodic potential is found to be 107 mV which suggests sluggish electron transfer and quasi reversible redox equilibrium, maintained by the FAD/FADH2 couple (Figure 4, blue dashed line). CV response of GOx@ZIF-8(AuNPs) composite shows immense faradaic behaviour with visible sharp peaks in both cathodic and anodic potential at Epa= - 408 mV and Epc= 480 mV, having a peak separation of ∆Ep= 72 mV, which is consistent with reported value of Epa= - 415 mV and Epc= - 473 mV,9 represents significantly high electron transfer rate of the reversible redox equilibrium of the FAD/FADH2 redox couple which can be demonstrated as the following redox reaction: GOx(FAD) + 2H+ +2e- ↔ GOx(FADH2)


(3)

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The incorporation of Au nanoparticle is thus playing an importance role, not only by shortening the electron tunnelling distance by mediating the charge but also increasing the sensitivity of the system upto 10-folds. The surface plasmon resonance energy of Au nanoparticle is so high that it can establish effective interaction with biomolecules resulting an immense increment in the output.40

Figure 4. Cyclic Voltammetry of ZIF-8@GOx (blue dashed line), Au@ZIF-8 (red dashed line) and GOx@ZIF-8(AuNPs)(Black line) at a scan rate of 0.1 V/S in N2 saturated 0.1M pH 7.4 PBS.

The effect of scan rate on the electrochemical response of GOx was evaluated and depicted in Figure 5. The scan rate measurement was also taken in N2 saturated 0.1 M pH 7.4 PBS buffer as an electrolyte. Both the anodic and cathodic current increases linearly with the increase of scan rate from 50 to 300 mV/S and is depicted in the inset of Figure 5. The result follows the Randles Sevcik equation, i.e. the peak current is linearly proportional to the square root of scan rate for a faradaic reaction when other parameters are kept constant. This suggests that the process is diffusion limiting. The extrapolation of fitted line towards the y axis does not meet at zero coordinate which conveys the presence of non-faradaic currents as well and attributed to the double layer across the electrode electrolyte interface. The result ACS Paragon Plus Environment


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also suggests that the FAD/FADH2 process is a surface-active process as there are very small shifts in current at the low and high scan rate.

Figure 5. Cyclic Voltammetry of GOx@ZIF-8(AuNPs) at different scan rates from 50 mV/S to 300 mV/S in N2 saturated 0.1M pH 7.4 PBS. Plot of peak current versus square root of scan rate is depicted inset. 3.4. Bio-electroactivity of the redox probe The bioactivity of GOx@ZIF-8(AuNPs) was determined by estimating electroactive protein density (ᴦ, mol cm-2). It could be estimated by integration of reduction peak at 100 mV/s by following the equation: Q = nFAᴦ

(4)

Where Q, n, F, A denotes their native electrochemical designation. The surface coverage for GOx is calculated to be 3.93 × 10-10 mol cm-2, which is quite superior to other reports.9 The possible reason for this high surface coverage is cavity size of ZIF-8 which accommodates GOx in its cavity. The electron transfer rate constant (ks) was evaluated from the separation of the peak potential according to the model suggested by Laviron.41 To calculate ks, the


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separation between the cathodic and anodic peak potentials (∆Ep) at different scan rates is utilized using following equations:

234

.567 8

67

9 (1 ) : log(1 ) (1 ) log – log ?8@AB – log CD E

(5)

Charge transfer coefficient is calculated by the following equation: 67

I8@A

34F GH – ?I8@B ln ? 67K B

(6)

L

67

34M GH – ?(NOI)8@B ln ?

(NOI)8@A 67KL

B

(7)

Where, Epc and Epa are the peak current in cathodic and anodic directions and other notations carry their usual meaning. Charge transfer coefficient (α) was calculated 0.413 from equation 6 and 7. A peak potential separation of 72 mV is considered at a scan rate of 100 mV/S. The rate constant of GOx at the GCE/ GOx@ZIF-8(AuNPs) is (2.59 ± 0.02) s−1, which is equivalent or rather higher than the values reported in the literature.42 This value suggests that the redox couple FAD/FADH2 is sufficiently redox active and probably can work in any kind of chemical and biological environment with a low detection limit. 3.5 Biosensor response of ZIF-8@GOx(AuNPs): High surface coverage, fast electron transfer and excellent redox stability of GOx@ZIF8(AuNPs) at -460 mV manifests it as an excellent candidate for fabrication of biosensor for low level detection of glucose from sweat and other body fluids. To get the idea about the activity of the composite towards real environment, CV was taken with air saturated 0.1 M pH 7.4 PBS buffer as electrolyte at a scan rate of 100 mV/S in the range of -0.2 to -0.7 V vs Ag/AgCl (sat. KCl) and compared with N2 saturated 0.1 M pH 7.4 PBS buffer. The result is depicted in Figure 6. The result exhibits an increase of the cathodic peak of air saturated PBS with respect to N2 saturated PBS whereas a more or less equal anodic peak was observed for both. The result can be explained by possible oxidation of reduced enzyme (GOx-FADH2) to ACS Paragon Plus Environment


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its oxidized form GOx-FAD by the dissolved oxygen and this causes the loss of reversibility of the catalytically regenerated enzyme to its oxidized form. The fact could be established strongly as we had repeated this experiment not by adding glucose but H2O2 and we found a characteristic increase of cathodic current while the successive addition of H2O2 at -0.45V vs Ag/AgCl (sat. KCl). The result is depicted in Figure 7. Mechanism of this process was rather interesting from other reported GOx and peroxidase-based glucose sensor. The cathodic current decreases due to the partial oxidation of hydrogen peroxide to oxygen occurring at the electrode electrolyte interface and the generated oxygen is getting dissolved in the peroxide solution. . The possible electrode reaction for this purpose will be expressed as follows: GOx -FAD + H2O2 → GOx-FADH2 + O2

(8)

The reaction is not unidirectional as after a certain time, the dissolved oxygen play the reverse role to generate again H2O2 by oxygen reduction reaction which leads to sluggish the forward reaction. The result can be seen in Figure 7 as after 120 seconds the change of current step is not so smooth as the competitive inhibition of O2 with H2O2.

Figure 6. Cyclic Voltammetry of GOx@ZIF-8(AuNPs)at a scan rates of 0.1 V/SACS of N saturated 0.1M pH 7.2 Paragon Plus Environment 2 and air

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Figure 7. Amperometric measurement of oxygen reduction reaction at -0.45 V vs Ag/AgCl (sat. KCl) with successive addition of H2O2 resulting a decrement in cathodic current.

3.6. Calibration plot We had explored the electroctalytic activity of GOx@ZIF-8(AuNPs) vividly and for this purpose we had done CV vs Ag/AgCl (sat. KCl) in air saturated PBS pH 7.4 buffer and the glucose concentration is increased from 0 to 8 mM. The decrease of cathodic peak current had been noticed and depicted in Figure 8 and it had been correlated with the concentration of glucose. A linear increment of anodic current has been found which depicts good catalytic activity of the composite towards glucose oxidation. The possible electrode reaction of glucose oxidation can be correlated by following equations: Glucose + GOx -FAD → Gluconic acid + GOx -FADH2


(9)


GOx -FADH2 + O2 → GOx -FAD+ H2O2

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(10)

Figure 8. Cyclic voltammogram of GOx@ZIF-8(AuNPs)with addition of glucose 2mM-8mM in air saturated 0.1 M PBS pH 7.4 buffer. Plot of current versus glucose concentration at cathodic peak is plotted inset The first step comprises the reduction of GOx-FAD to GOx-FADH2 resulting glucose to form gluconic acid. In second step, oxygen reduction reaction occurs by which natural oxygen converted to H202 which leads to increment of cathodic peak current. The conventional amperometry (potentiostatic) experiment had performed by successive addition of 0.1 mM glucose to the air saturated pH 7.4 buffer solution at a characteristic potential of – 0.45 V vs Ag/AgCl (sat. KCl) (Figure 9). Dynamic stairs of current with quick steady state had been observed upon successive addition of glucose due to the formation of hydrogen peroxide. %RSD was calculated 1.2% for 20 successive dilutions, depicting the stability of the composite catalyst. The steady state current had been plotted with the concentration added and a linear plot had been found, depicted in the inset of Figure 9 having a detection limit of 50 nM of glucose. The current achieved steady state gradually after 20


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successive dilution due to the increment of dissolved oxygen which disturbs the equilibrium of FAD/FADH2. This low detection limit can be attributed to our claim of measuring blood glucose from sweat which encourages non-invasive, point of the care device.

Figure 9. Typical steady state amperometric measurement of catalytic glucose oxidation at -0.45 V vs Ag/AgCl (sat. KCl) with successive addition of 0.1 mM glucose. Calibration plot of concentration versus (full and linear region) current has been depicted inset. 3.7 Shelf-life: The probe has been stored in powder form in air sealed container at room temperature and used in sensor application after 120 days. The composite was found extremely stable even after 120 days of storage. The cyclic voltammetry has been performed under the same condition as discussed above. The Cyclic voltammetry plot is depicted in Figure S11 (See ESI). A negligible change in peak position (

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