Designation of biosensor detecting bacteria in refrigerator
Overview
Although refrigerators can extend the shelf life of food, they are not sterile environments. Under low temperature conditions, some bacteria such as Shigella, Salmonella, Yersinia, etc. can still survive and even reproduce (Fig.1). Once these bacteria contaminate food which are ate by people, they may cause diseases such as food poisoning, acute gastroenteritis, and even death in severe cases.
Fig.1 Bacteria that could contaminate the food in refrigerator.
We have designed an efficient and sensitive biosensor that can monitor pathogenic bacteria in the refrigerator at actual time, reminding people to clean the refrigerator in a timely manner, and ensuring food safety and avoiding illness.
The sensor is designed based on aptamer (nucleic acid aptamer) recognition technology, combined with toehold switch detection strategy, and uses cell-free system and hydrogel materials to detect refrigerator bacteria. At the same time, using RGB sensors and integrated hardware systems, the detected color signals are converted into electrical signals and displayed on the refrigerator door with LED lights.
1. Introduction to Aptamer
Aptamer biosensor is a novel biosensor based on aptamer as a biosensing element. Aptamer is a nucleic acid sequence that can bind to various target molecules with high affinity and specificity. Aptamer biosensors utilize the specific binding between aptamers and targets (such as bacteria) to convert biometric results into measurable signals for the detection of targets. Aptamer has the following characteristics:
(1) High specificity: Aptamers can accurately recognize and bind to specific target molecules, such as specific proteins or DNA/RNA sequences on bacterial surfaces. We have identified Aptamers that can recognize and bind to common pathogenic bacteria in refrigerators, such as Salmonella, Shigella, and Escherichia coli, through literature review, and used them to construct biosensors (Table 1).
Table 1 Aptamer sequences of Shigella, Salmonella and E. coli.
(2) Good stability: Compared with antibodies, aptamers have higher thermal and chemical stability, and can maintain activity for a long time in low-temperature and humid environments of refrigerators.
(3) Easy to synthesize and modify: Aptamers can be quickly obtained through chemical synthesis methods and are easy to functionalize, such as connecting fluorescent groups, biotin, etc., for the purpose of detecting signal output.
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2. Introduction to Toehold Switch
Toehold Switch is a detection strategy based on RNA structural changes, which consists of two main components: Switch RNA and Trigger RNA (Fig.2). Switch RNA has a special hairpin structure, where the ribosome binding site (RBS) is located on the loop of the hairpin structure, and the initiation codon (AUG) is located in the stem region. Trigger RNA can form complementary pairing with specific regions of Switch RNA, thereby opening the hairpin structure, exposing RBS, and initiating the expression of downstream reporter genes, achieving rapid and accurate detection of target molecules.
Fig.2 The principle of RNA Toehold Switch.
The toehold switch technology has high specificity and sensitivity, and can accurately identify target molecules, including pathogenic bacteria, in complex biological environments. We will combine aptamer with toehold switch to construct a highly sensitive detection system. When the target bacteria are present, aptamers bind to them and trigger conformational changes in the toehold switch, leading to the expression of the reporter gene.
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3. Cell free system hydrogel
Cell free system is based on the rapid expression of proteins in vitro using cell extracts (Fig.3). This system bypasses processes such as bacterial transformation, clone screening, and cell lysis, enabling more precise and convenient control of reaction substrates, reducing the impact of bacteria on protein production, and possessing advantages such as multi-functionality and flexibility. The cell-free system mainly consists of cell extracts (including ribosomes, RNA polymerases, and other transcription and translation auxiliary proteins), a mixture of complementary cofactors for protein synthesis (such as amino acids, peptides, energy, metabolic cofactors, etc.), and DNA templates. Cell free systems are of great significance in the development of biosensors, as they can quickly express target molecules for detection.
Fig.3 The flow chart of preparing for cell free system
In the design of biosensors, hydrogels can be used as carrier materials to fix detection elements, maintain the stability of bioactive molecules, and provide a suitable detection environment. In our project, hydrogels are used as carriers and stabilizers of cell-free systems to maintain the activity and stability of cell-free systems (Fig.4). At the same time, the porous structure of hydrogel is conducive to the exchange of nutrients and the emission of metabolites, thus improving the efficiency of detection.
Fig.4 Bacteria are detected by aptamer toehold switch on hydrogel containing cell free system.
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4. Detector Hardware
Detector Hardware is composed of 4 parts: cell free system hydrogel, main board, RGB sensor and a breadboard with pilot LED lamp.
When the bacteria in the refrigerator fall on the hydrogel made by the cell-free system, they will combine with the specific aptamer, trigger the toehold switch to open, and cause the expression of the reporter gene. The expression product of the reported gene is galactosidase, which can catalyze the conversion of substrates into blue products. The blue products is recognized by RGB sensors and converts the color signal into an electrical signal, which is transmitted to the indicator light through the motherboard, causing the LED red light to light up, reminding people to clean the refrigerator (Fig.5).
Fig.5 The detector is composed of cell free system hydrogel, main board, RGB sensor and a breadboard with pilot LED lamp.
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5. Summary
The biosensor combines the advantages of aptamer recognition technology, toehold switch detection strategy, cell-free system and hydrogel technology, and can realize efficient, sensitive and real-time monitoring of pathogenic bacteria in refrigerators. Through an integrated hardware system, this sensor can be conveniently applied to food safety monitoring in places such as homes, restaurants, and supermarkets.
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References
[1] Duan N, Ding X, Wu S, et al. In vitro selection of a DNA aptamer targeted against Shigella dysenteriae. J Microbiol Methods. 2013;94(3):170-174. doi:10.1016/j.mimet.2013.06.016
[2] Dua P, Ren S, Lee SW, et al. Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats. Mol Cells. 2016;39(11):807-813. doi:10.14348/molcells.2016.0167
[3] Pan Q, Zhang XL, Wu HY, et al. Aptamers that preferentially bind type IVB pili and inhibit human monocytic-cell invasion by Salmonella enterica serovar typhi. Antimicrob Agents Chemother. 2005;49(10):4052-4060. doi:10.1128/AAC.49.10.4052-4060.2005
[4] Wang GA, Wu X, Chen F, Shen C, Yang Q, Li F. Toehold-Exchange-Based Activation of Aptamer Switches Enables High Thermal Robustness and Programmability. J Am Chem Soc. 2023;145(5):2750-2753. doi:10.1021/jacs.2c10928
[5] Li H, Tang Y, Li B. Homogeneous and Universal Detection of Various Targets with a Dual-Step Transduced Toehold Switch Sensor. Chembiochem. 2020;21(10):1418-1422. doi:10.1002/cbic.201900749
[6] Zheng H, GhavamiNejad A, GhavamiNejad P, Samarikhalaj M, Giacca A, Poudineh M. Hydrogel Microneedle-Assisted Assay Integrating Aptamer Probes and Fluorescence Detection for Reagentless Biomarker Quantification. ACS Sens. 2022;7(8):2387-2399. doi:10.1021/acssensors.2c01033
[7] Tang J, Jia X, Li Q, et al. A DNA-based hydrogel for exosome separation and biomedical applications. Proc Natl Acad Sci U S A. 2023;120(28):e2303822120. doi:10.1073/pnas.2303822120
[8] Kato S, Ishiba Y, Takinoue M, Onoe H. Histamine-Responsive Hydrogel Biosensors Based on Aptamer Recognition and DNA-Driven Swelling Hydrogels. ACS Appl Bio Mater. 2024;7(6):4093-4101. doi:10.1021/acsabm.4c00423
[9] Liu M, Zhang J, Liu S, Li B. A label-free visual aptasensor for zearalenone detection based on target-responsive aptamer-cross-linked hydrogel and color change of gold nanoparticles. Food Chem. 2022;389:133078. doi:10.1016/j.foodchem.2022.133078
[10] Di Y, Wang P, Li C, et al. Design, Bioanalytical, and Biomedical Applications of Aptamer-Based Hydrogels. Front Med (Lausanne). 2020;7:456. Published 2020 Oct 22. doi:10.3389/fmed.2020.00456
[11] Sánchez-Costa M, Urigoitia A, Comino N, et al. In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial. ACS Appl Mater Interfaces. 2024;16(13):15993-16002. doi:10.1021/acsami.4c01388
[12] Li Y, Bi X, Wu M, et al. Adjusting the stiffness of a cell-free hydrogel system based on tissue-specific extracellular matrix to optimize adipose tissue regeneration. Burns Trauma. 2023;11:tkad002. Published 2023 Mar 1. doi:10.1093/burnst/tkad002
[13] Chen J, Zhuang X, Zheng J, et al. Aptamer-based cell-free detection system to detect target protein [published correction appears in Synth Syst Biotechnol. 2023 May 16;8(2):339. doi: 10.1016/j.synbio.2023.05.001]. Synth Syst Biotechnol. 2021;6(3):209-215. Published 2021 Aug 13. doi:10.1016/j.synbio.2021.07.004