Elsevier

Bioelectrochemistry

Volume 142, December 2021, 107919
Bioelectrochemistry

A biomimetic “intestinal microvillus” cell sensor based on 3D bioprinting for the detection of wheat allergen gliadin

https://doi.org/10.1016/j.bioelechem.2021.107919Get rights and content

Highlights

  • A 3D-bioprinting electrochemical cell sensor for assessment of wheat gliadin is presented.

  • FCONp/ MWCNT-CDH/ gelatin methacryloyl composite hydrogel was constructed.

  • A biomimetic intestinal microvilli structure was designed, printed and applied to immobilize RBL mast cells.

  • The study provides an alternative to traditional allergen detection methods.

Abstract

A biomimetic “intestinal microvillus” electrochemical cell sensor based on three-dimensional (3D) bioprinting was developed, which can specifically and accurately detect wheat gliadin. Self-assembled flower-like copper oxide nanoparticles (FCONp) and hydrazide-functionalized multiwalled carbon nanotubes (MWCNT-CDH) were innovatively synthesized to improve the sensor performance. A conductive biocomposite hydrogel (bioink) was prepared by mixing FCONp and MWCNT-CDH based on GelMA gel. The cluster-shaped microvillus structure of small intestine was accurately printed on the screen printing electrode with the prepared bioink using stereolithography 3D-bioprinting technology, and then the Rat Basophilic Leukemia cells were immobilized on the gel skeleton. Next, the developed cell sensor was used to effectively detect wheat allergen gliadin. The experimental results show that the bioprinted cell sensor sensitively detects wheat gliadin when the optimized cell numbers and immobilized time are 1 × 106 cells/mL and 10 min, respectively. The linear detection range is 0.1–0.8 ng/mL, and the detection limit is 0.036 ng/mL. The electrochemical cell sensor based on 3D printing technology has excellent stability and reproducibility. Thus, a simple and novel electrochemical detection approach for food allergens was established in this study with potential application in food safety detection and evaluation.

Introduction

In recent years, the number of food allergy patients is increasing, and the incidence of allergy is also increasing year by year [1]. Food allergy has become a severe food safety problem. In daily life, milk, peanuts, wheat, and fish are the most common food allergens [2], [3]. So far, a few treatments are available for food allergies, among them, AR101 oral immunotherapy treatment led to rapid desensitization to peanut protein, with a predictable safety profile that improved with treatment. But avoiding any food containing allergens is more crucial for food allergy-related public concerns [4]. Therefore, rapid detection and early warning of food allergens have great significance [5]. Gluten from wheat can cause allergic reactions, and gliadin is its main component [6]. As a small amount of allergen in a food, gliadin can cause severe immune reactions, eventually leading to chronic inflammation and damage in the upper small intestine. Therefore, sensitive and quantitative detection of gliadin has become vital, and it is essential to develop a highly sensitive quantitative detection method for wheat allergens. So far, the most mature allergen detection method is based on immune recognition (such as ELISA method) [7], but it has some disadvantages such as strict operating process, high reagent cost, and time consuming [8]. Therefore, it is important to develop an alternative method. Recently, electrochemical cell sensor development is a new direction of biosensor research [9]. With the rapid development of microelectronic technology and in vitro culture technique, construction of cell sensors using living cells as a sensing medium has been widely used in food hazards, environmental toxicity [10], other toxic materials testing, drug evaluation [11], and many other fields.

An ordinary cell sensor mainly relies on a two-dimensional (2D) cell immobilization scheme [12], but the 2D cell culture environment is just a simple accumulation of cells to form a spatial dislocation distinct from the human microenvironment, and an in vitro tissue model suffers from high cost, long cycle, and low repetition rate. Previous studies were conducted on only mast cells, 2D or mixed with a gel after the 3D state was fixed on the surface of sensing interface (electrode) [13]. The cells were simply accumulated without a stereoscopic simulation of the organization structure [14], [15], and they cannot fully simulate the allergen reaction mechanism in the body and lack the construction characteristics of primary organs and thus unable to produce a higher level of functional characteristics. True allergic reactions occur in vivo; the mast cells are attached to the free surface of tissue and organ degranulation [16]. After being ingested and digested, allergen proteins first enter the intestine for absorption, so the small intestine is the primary organ for allergic reaction. However, there are still challenges with existing technologies. Bioprinting cannot really simulate the tissue or organ of natural physiological function.

With the development of new materials and equipment, three-dimensional (3D) printing, a rapidly developing emerging technology [17], has become a research hotspot in recent years, and it has been widely used in various fields [18], [19] such as drug analysis, cell tissue culture, and environment. The technology has unique advantages such as preparation of sample models with multilevel fast detection ability, mainly in the wide application of biological medicines, such as 3D printing support for autogenous bone particles, 3D printing of softening blood vessels, and 3D coculture tumor model for drug toxicity analysis, but fewer in the field of biosensor applications [20], especially in the application of electrochemical cell sensing direction [21]. Currently, cell immobilization scheme and high-throughput preparation are the key to building cell sensors, and 3D printing technology would effectively solve these problems. Flexible printing design provides more possibilities for sensing direction [22]. If the technology can be used quickly and accurately for the preparation of a group of hundreds of sensors, it can greatly reduce the error caused by human operation and preparation cost, widen the scope of cell sensing application, and improve the stability of sensor between different batches [23], [24].

3D printing technology was used to prepare a small intestinal structure of microvillus in macroscale by adjusting the organizational size and structure, conducted the adhesion of mast cells in the intestinal structure [25], [26] and allergic simulation in a real environment to achieve 3D vertical detection. Thus, we tried to achieve single cell detection by tissue engineering, making it possible for 3D organizational detection and more real and more comprehensive biosensor simulation.

We selected methyl acryloyl gelatin (GelMA hydrogel) printing ink [27], because it contains GelMA for the modification of the double bond of gelatin. The solution can be cured using a UV-curable gel. GelMA molecules contain the RGD sequence of cell adhesion [28]. The 3D network structure has excellent biocompatibility and can be formed with micro and nanoporous hydrogel stents to load cells [28]. However, pure GelMA has no electrical conductivity, and it is unable to better simulate biological tissues in a weak conductive environment [29]. The cells cannot communicate electrical signals with each other. Therefore, we need new conductive materials to improve the detection sensitivity without affecting the cell activity. Currently, several methods are available to improve the conductivity, such as by adding conductive materials including metal nanoparticles, graphene oxide, carbon nanotubes (CNTs), and conductive polymers. Graphene-based carbon nanomaterials are the most commonly used conductive materials [30]. However, the agglomeration of nanofillers and large particle size resulted in poor printability. Polyaniline (PANi) and polypyrrole (PPy) have good application prospects, but they are insoluble in water. Therefore, it is essential to develop conductive materials that can overcome the above defects. Hydrazide-functionalized multiwalled CNTs that can be stably dispersed in water and work in harmony with gels [31]. These positively charged nanotubes theoretically interact with negatively charged cell membranes to enhance their function by improving cell adhesion. However, the use of a single material does not provide significant signal amplification. Composite nanomaterials have good antimicrobial properties and high electrocatalytic ability. Therefore, they have recently attracted much attention [32], [33], especially copper nanomaterials play an important role in the preparation of sensors, generally with a metal compound [34]. However, gold and silver, the main representative metals, suffer from high cost. Copper nanomaterials, in contrast, are inexpensive and easy to synthesize [35]; they can be used to build more advanced 3D nanosystems [36]. Thus, flower copper oxide nanoparticles (FCONp) were synthesized for the preparation of high conductivity electrochemical sensors.

Mast cells expresses a large array of Fc receptors on their surface, including Fc∊RI, FcγRI, and FcγRIII, which could selectively bind murine IgE or IgG antibodies so that the subsequent cross-link with an allergen would trigger cellular degranulation. According to immunity rule, sensitized mast cells can selectively bind with wheat allergen protein within minutes. This specific recognition process causes a series of intracellular activities, including cellular degranulation and cellular content release, such as histamine, tryptase and β-hexosaminidase. Then, changes in the biochemical properties of these mast cells, which immobilized between the conducting surfaces, may affect their capacitance, allowing for the real-time monitoring of mast cell sensitization levels. Therefore, the quantitative detection of allergen protein can be indirectly achieved by evaluating the degree of mast cell degranulation or cellular content release [37].

In this study, a new electrochemical mast cell sensor based on 3D bioprinting technology was developed, which can specifically and accurately detect wheat gliadin. To improve the sensitivity and biocompatibility of the sensor, self-assembled flower-like copper oxide nanosheets (FCONp) and hydrazide-functionalized multiwalled CNTs (MWCNT-CDH) were synthesized for the first time to prepare an electroconductive biohybrid hydrogel. Stereolithography 3D-bioprinting technology can be used for the high-throughput preparation of cell sensors. The microvillus structure of small intestine was printed to imitate human small intestinal villus tissues. Thus, the gliadin antibody-sensitized mast cells were immobilized on the screen printing electrode through the biological affinity of the 3D-printed small intestinal microvilli. The experimental results of wheat gliadin cell sensor detection show that the cell sensor not only has a good linear concentration range, excellent stability, and reproducibility, can greatly shorten the cell sensor build time, and reduce the cost of preparation. The electrochemical results were compared with the results of traditional analysis methods. This simple and inexpensive cell sensor can be used for rapid and simple electrochemical detection of wheat allergens.

Section snippets

Materials and apparatus

Wheat gliadin standard was purchased from sigma Biological Sigma-Aldrich Inc. (St. Louis, MO, USA). Anti-Gliadin antibody 14D5 was purchased from Abcam (Cambridge, MA, USA). Carboxylated multi-walled carbon nanotubes were obtained from Xian Feng Nanomaterials Technology Co., Ltd. (Nanjing, China). Copper chloride dihydrate, N-(3-diethylaminopropyl)-N’-ethylcarbodiimide (EDC), carbodihydrazide and hydroxyl-benzotriazole were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Rat basophilic

Construction and working principle of the sensor

After being ingested and digested, allergen proteins first enter the intestine for absorption, so the small intestine is the primary organ for allergic reaction. Therefore, we used 3D printing technology to prepare a small intestinal structure of microvillus in macroscale by adjusting the organizational size and structure and conducted the adhesion of mast cells in the intestinal structure. In this way, the cells do not undergo a simple accumulation. It is crucial that allergen protein

Conclusions

In this study, 3D bioprinting technology was used to develop a simple and novel cell sensor. A small intestinal villi structure was printed, which simulates human small intestinal villi tissues. A cluster type intestinal microvillus was performed on a disposable screen printing electrode and adhered to RBL mast cells, thus building a new generation of biomimetic micro organization of electrochemical cell sensors. This sensor was used for the effective detection of wheat gliadin. This sensor has

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work has been supported by The National Key Research and Development Program of China (2020YFC1606801), Natural Science Foundation of Jiangsu Province (BK20180160), Postgraduate Research Practice Innovation Program of Jiang -su Province (No.KYCX20_1340), Special Program of the State Administration for Market Regulation (2019YJ047), Science and Technology Program of Nanjing Administration for Market Regulation (Kj2019042).

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