Elsevier

Biomaterials

Volume 237, April 2020, 119782
Biomaterials

Transdermal colorimetric patch for hyperglycemia sensing in diabetic mice

https://doi.org/10.1016/j.biomaterials.2020.119782Get rights and content

Abstract

The integration of sampling and instant metabolite readout can fundamentally elevate patient compliance. To circumvent the need for complex in-lab apparatus, here, an all-in-one sampling and display transdermal colorimetric microneedle patch was developed for sensing hyperglycemia in mice. The coloration of 3,3′,5,5′-tetramethylbenzidine (TMB) is triggered by the cascade enzymatic reactions of glucose oxidase (GOx) and horseradish peroxidase (HRP) at abnormally high glucose levels. The HRP in the upper layer is biomineralized with calcium phosphate (CaP) shell to add a pH responsive feature for increased sensitivity as well as protection from nonspecific reactions. This colorimetric sensor achieved minimally invasive extraction of the interstitial fluid from mice and converted glucose level to a visible color change promptly. Quantitative red green and blue (RGB) information could be obtained through a scanned image of the microneedle. This costless, portable colorimetric sensor could potentially detect daily glucose levels without blood drawing procedures.

Introduction

Diabetes mellitus refers to an incurable chronic disease characterized by abnormally high blood-glucose levels, known as hyperglycemia [1]. Diagnostic devices for home-based daily monitoring of glucose level can aid diabetes patients in seeking timely treatments to prevent symptoms including nephropathy, high blood pressure, and stroke [2]. Minimally invasive sample collecting modules and diagnostic information-display modules are two fundamental factors for developing a portable, cheap and simple to use glucose sensor.

The existing glucose level measurements require invasive blood sampling [3]. As a painless alternative, strategies using a microneedle array patch have been extensively adopted for extracting [4,5] and detecting metabolites [6] or delivering therapeutic drugs [[7], [8], [9]] in the interstitial fluid, which is the extracellular fluid surrounding tissue cells that possesses similar composition of clinically important biomarkers to blood, including glucose levels [10].

Colorimetric bio-sensing is favorable for on-site analysis and point-of-care diagnosis, without the need for complex in-lab apparatus [11,12]. Integration of a dual enzymatic system that consists of glucose oxidase and peroxidase could achieve rapid colorimetric sensing of glucose [13]. Glucose oxidase [14,15] is responsible for transforming glucose into chemical signals of gluconic acid and H2O2. Peroxidase [[16], [17], [18]], thereafter, can catalyze substrates including 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), and diazoaminobenzene (DAB) to generate color change visible to the naked eye in the presence of hydrogen peroxide (H2O2). However, enzymatic reactions are sensitive to environmental changes such as pH and digestive proteases. In this case, the byproduct gluconic acid would significantly lower the surrounding pH and inhibit HRP activity with the increase of glucose [19], which greatly decreases the sensitivity of the system. In addition, current colorimetric sensors are mainly dependent on in-solution reactions, which is a major issue for storage and transportation.

Herein, we describe a poly(vinyl alcohol) (PVA)-based double layer microneedle patch, achieving both in situ dermal sample collection and instant color display. The bottom needle layer is embedded with glucose oxidase, while the upper layer is immobilized with biomineralized HRP (calcium phosphate-encapsulated HRP, HRP-CaP) and TMB. Upon glucose challenge, glucose oxidase selectively converts glucose into gluconic acid and H2O2, thereby lowering the local pH to free HRP from HRP-CaP and induce the catalytic oxidation of the TMB substrate in the presence of hydrogen peroxide (Fig. 1a). It features a dual responsive mechanism to increase sensitivity accompanied by a layer of protection from nonspecific oxidation. The incorporation of a biocompatible inorganic shell maintained the intrinsic catalytic efficiency of the natural enzymes with improved robustness. The costless (5 cents for each test) microneedle sensor could distinguish the glucose level between 50 mg/dL and 400 mg/dL within 30 s in vitro and the hyperglycemia occurrence in mice could be recognized with the naked eye within 4 min after insertion.

Section snippets

Preparation of biomineralized HRP formulation

Briefly, 1 mg of HRP was dissolved directly in 1 mL of DMEM medium and kept for three days or longer to reach equilibrium. An additional 3.2 μL of CaCl2 (1 M) was introduced to DMEM (with 1.8 mM Ca2+) to reach a final Ca2+ concentration of 5 mM and was incubated at 37 °C under 5% CO2 for 24 h. The reaction tubes were sealed with aluminum foil with holes to adjust the system-environment substrate exchange. The HRP-CaP particles was purified from DMEM and washed three times with water by

Synthesis and characterization of biomineralized HRP (HRP-CaP)

Like many biomineralization-related proteins, HRP contains Ca2+ binding ligands that serve as “nucleation sites” for concentrating cationic calcium ions and generate tiny crystals around the peptides. Eventually, the crystals assemble into spherical particles [18,20]. The mild biomineralization process is conducted in the Dulbecco's modified Eagle's medium (DMEM) containing an optimal Ca2+ concentration of 5 mM for 24 h at 37 °C with 5% CO2 (Fig. S1). The size shift from 6.2 ± 1.4 nm to

Conclusions

In summary, we have presented a glucose colorimetric sensor capable of in situ detection of hyperglycemia in mice. Sampling and result display are two important features for practical applications of portable glucose sensors. The double layer microneedle array patch designed with GOx/HRP dual enzyme systems for glucose sensing and color conversion has the potential to achieve both attributes simultaneously. The biomineralization design of HRP takes advantage of the two products from the

Declaration of competing interest

The authors declare no conflict of interest.

Author contributions

Z.W. and H.L. contributed equally. Z.W. and Z.G. designed the experiments. Z.W., H.L., J.W., Z.C., G.C., D.W. and A.C. performed experiments and collected data. Z.W. and Z.G. analyzed the data and wrote the paper.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.

Acknowledgements

This work was supported by the grants from National Science Foundation (grant no.1708620), American Diabetes Association (grant no. 1-15-ACE-21), and grant from the start-up packages of UCLA.

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    Z.W. and H.L. contributed equally.

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