Cancer biosensing has been a hot topic in biomedical engineering in recent decades. One of the most important aspects of detection is to do it early in order to gain a time window within which an effective treatment course can be followed. Since the idea is well-appreciated, there have been many implementations of applications on all levels of the scientific community. From iGEM teams to Ph.D. projects, a lot has been researched about using bacteria for biosensing. Although many projects reached their initial goals of detection, the genetic system’s efficacy has remained generally unchanged and evidently insufficient.
General synthetic biosensors are inspired from biochemical computing systems . In such devices, chemicals act as an input to the tightly regulated pathways of proteins which then respond by providing a chemical / protein signal as an output. While such systems are easier to engineer, a major drawback lies in their unreliable half-life. In fact, the tighter the regulation, the better the output, and the faster the output decay of the system . This can be fundamentally improved by taking advantage of the metal respiratory system (Mtr), also known as Mtr oxidative pathway. In bacteria which maintain such systems, protein is no longer an object of detection, but their activity. Instead of overproducing a certain kind of likely toxic compound, one would only need to provide a target of electron transfer for these proteins (electrode), on which each protein will independently aid in electricity generation, as a part of their natural activity. Importantly, these proteins are normally recovered, relieving the necessity of overproduction. As such, Mtr oxidative pathway can be engineered to serve as an output for the signal of interest, by regulating protein production . This should result in an evolutionarily stable system in which continuous bacterial evolution should not exert a limiting effect over the engineered system. Hence, chemicals can act as an actuator of biocomputing, resulting in electricity propagation, which is otherwise known as bioelectric computing.
Bioelectric computers have been successfully employed in ecological toxicity sensors where fuel cell devices are poised to detect chemicals in a highly specific manner . These systems advance general biochemical computation by translating chemical signals into electricity and could serve as a paradigm shift from chemical to computer-like systems. Notably, such systems have been observed to be operational for up to 5 years . In addition, compared to traditional biosensors, which for example detect antibiotics in 24–48 hours, a bioelectric computer was able to measure the same antibiotic in 2-4 hours . More importantly, microbial fuel cells (MFCs) detection capacity can be restored without external manipulation, potentially replacing polluting single-use sensors with fully recyclable biosensors.
Synthetic biology is about introducing engineering principles to the life systems for a specific purpose. For our project we have devised to help aid cancer detection by advancing the existing testing systems to a more flexible, diverse and internet of things (IOT) application. While biosensors developed by microbial fuel cell apparatus are ubiquitous, most of the applications are dealing with highly sophisticated and less mobile devices. One of the greatest limitations of generic MFCs is the choice of anaerobic bacteria, which necessitates specialized sealing design. Such composition is difficult to redesign, given a different target is to be sensed. Moreover, operating expertise of anaerobic chambers greatly restricts end-user diversity.
We have aimed to create a proof of concept (POC) that would alleviate the aforementioned limitations and hence deliver a statement that MFC systems can be used for diverse applications. To achieve this, we have introduced an aerobic system with user-friendly apparatus that can be rebuilt using simple nut & bolt seals. In addition, we have eliminated in situ expertise requisite, by directly associating bacterial sensing to the Arduino IOT hardware. Our design makes it possible for a detection and data analysis to be performed at distances covered by the internet, which reasserts potential end-user diversification. Furthermore, we have integrated safety features which can guarantee at POC level, that the system malfunction is monitored and the local measures are taken. On top of all, we have built our sensing capacity over toehold switch, which is easy to engineer and grants versatility to the range of cancers or other targets to detect. While maintaining non-toxic bacteria for introducing synthetic circuits. Above all, our MFC was built using compostable PLA material and was controlled by reusable, versatile, cheap Arduino modules. As a result, if implemented at a wider scale, our MFC can overcome equity issues observed in systems that rely on expensive and rare technologies.
We focused on the cancer biomarker PANTR1, a long non-coding RNA (lncRNA) overexpressed in different types of cancer cells from the early stage, that was shown to promote cell migration, invasion, angiogenesis and proliferation and to repress apoptosis [7–15]. In our design, PANTR1 sensing, leads to heterologous pathway activation, which releases electrons, to be sensed by Arduino-based hardware.
Before leading to an applicable device, we have aimed to build a proof of concept (POC), which would be able to detect PANTR1, and more generally RNA cancer biomarkers, using toehold switches . After detection occurs, the biosensor pathway regulation is altered, which leads to the change of the produced current (Fig. 1 - Step 1). Change in current will be then recorded using the Arduino current sensor, which will be fed electrical information, through wires that are connected to the interconnection between anode and the cathode of the MFC (Fig. 1 - Step 2). Finally, using other modules, recorded analog output will be transmitted to the computer, where analysis will be performed (Fig. 1 - Step 4). Depending on the analysis report, if target detection is verified, feedback will be sent back to the arduino (Fig. 1 - Step 5).
Once the whole POC is built and deemed functioning, we will be able to move on to developing more appropriate output generating bacterias, and sensors. This will make the system modular, in which one will be able to replace the sensor, while hardware will remain functional for a variety of applications, expanding on the objective of reusability of electronics and creating a foundation for a more diverse device. Importantly, minimization will further shorten detection times to a few minutes . Such systems could make cancer testing cheaper, faster, and most importantly, versatile and convenient.
References Windmiller JR, Windmiller JR. Molecular scale biocomputing : an enzyme logic approach. (2012). https://escholarship.org/uc/item/519217jp (accessed June 6, 2022).
 Myers, Chris J. Engineering genetic circuits, 1st Edition. Boca Raton, FL: CRC Press (2010).
 Coursolle D, Baron DB, Bond DR, Gralnick JA. The Mtr respiratory pathway is essential for reducing flavins and electrodes in Shewanella oneidensis. Journal of Bacteriology (2010) 192: 467–474.
 Cui Y, Lai B, Tang X. Microbial Fuel Cell-Based Biosensors. Biosensors (2019) 9: E92.
 Kim BH, Chang IS, Gil GC, Park HS, Kim HJ. Novel BOD (biological oxygen demand) sensor using mediator-less microbial fuel cell. Biotechnology Letters (2003) 25: 541–545.
 Schneider G, Czeller M, Rostás V, Kovács T. Microbial fuel cell-based diagnostic platform to reveal antibacterial effect of beta-lactam antibiotics. Enzyme and Microbial Technology (2015) 73–74: 59–64.
 Vance KW, Ponting CP. Transcriptional regulatory functions of nuclear long noncoding RNAs. Trends in genetics: TIG (2014) 30: 348–355.
 Li W, Zheng J, Deng J, You Y, Wu H, Li N, Lu J, Zhou Y. Increased levels of the long intergenic non-protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells. Gastroenterology (2014) 146: 1714-1726.e5.
 Guo H, Wu L, Yang Q, Ye M, Zhu X. Functional linc-POU3F3 is overexpressed and contributes to tumorigenesis in glioma. Gene (2015) 554: 114–119.
 Tong Y-S, Wang X-W, Zhou X-L, Liu Z-H, Yang T-X, Shi W-H, Xie H-W, Lv J, Wu Q-Q, Cao X-F. Identification of the long non-coding RNA POU3F3 in plasma as a novel biomarker for diagnosis of esophageal squamous cell carcinoma. Molecular Cancer (2015) 14: 3.
 Shan T-D, Xu J-H, Yu T, Li J-Y, Zhao L-N, Ouyang H, Luo S, Lu X-J, Huang C-Z, Lan Q-S, Zhong W, Chen Q-K. Knockdown of linc-POU3F3 suppresses the proliferation, apoptosis, and migration resistance of colorectal cancer. Oncotarget (2016) 7: 961–975.
 Lang H-L, Hu G-W, Chen Y, Liu Y, Tu W, Lu Y-M, Wu L, Xu G-H. Glioma cells promote angiogenesis through the release of exosomes containing long non-coding RNA POU3F3. European Review for Medical and Pharmacological Sciences (2017) 21: 959–972.
 Li Y, Li Y, Wang D, Meng Q. Linc-POU3F3 is overexpressed in hepatocellular carcinoma and regulates cell proliferation, migration and invasion. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie (2018) 105: 683–689.
 Wan X, Xiang J, Zhang Q, Bian C. Long noncoding RNA POU3F3 promotes cancer cell proliferation in prostate carcinoma by upregulating rho-associated protein kinase 1. Journal of Cellular Biochemistry (2019) 120: 8195–8200.
 Li W, Wu X, She W. LncRNA POU3F3 promotes cancer cell migration and invasion in nasopharyngeal carcinoma by up-regulating TGF-β1. Bioscience Reports (2019) 39: BSR20181632.
 Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159: 925–939.
 Moon H, Chang IS, Kang KH, Jang JK, Kim BH. Improving the dynamic response of a mediator-less microbial fuel cell as a biochemical oxygen demand (BOD) sensor. Biotechnology Letters (2004) 26: 1717–1721.
 Logan BE. Exoelectrogenic bacteria that power microbial fuel cells. Nature Reviews. Microbiology (2009) 7: 375–381.