Background

Biosensors are analytical devices that elicit a measurable signal in response to specific biological processes or the presence of a molecule. Using these tools, scientists have made crucial breakthroughs in disease diagnosis, environmental monitoring, food control, drug detection, forensics, and biotechnology [1]. Developing biosensors is also the first step in degrading toxic chemical compounds. In a laboratory setting, researchers need to be able to measure changes in the concentration of a molecule over time in order to determine whether a specific strategy is useful to degrade that molecule. As a result, biosensors have become the backbone of synthetic biology [2].

An ideal biosensor will elicit a response proportional to the amount of analyte present. The analyte is the substance of interest the biosensor is trying to detect. When the bioreceptor, a specific enzyme, cell, or DNA sequence, recognizes the analyte, it will work with the transducer to create a measurable response [1]. Specifically, biosensors rely on promoters to control their responses. Promoters are DNA sequences that regulate when and how strongly a gene is expressed. Bacteria are often modified to produce Green Fluorescent Protein, or GFP, in the presence of an analyte. This protein creates a fluorescent signal that can be measured through a plate reader to determine the amount of the molecule [3].

Biosensor Example

Figure 1. Example of a biosensor for drug screening processing used a red fluorescent reporter [2]


A relatively recent paper from 2006 by the Weizmann Institute of Science presents a comprehensive library of about 2000 fluorescent transcriptional promoters for Escherichia coli K12, a well-studied gram-negative bacteria commonly used for biosensor development. Several E. coli strains are also nonpathogenic [4]. The library covers the majority of promoters in the organism and is expressed from a low-copy plasmid. This presents an opportunity to develop a multitude of different biosensors for molecules compatible with the promoters [4]. Therefore, the goal of our project is to identify which promoters produce the ideal levels of fluorescence for several different molecules as a starting point for future biosensor development.

We chose ten molecules for our screening that cover a wide range of topics, from environmental pollution to healthcare tools. Biosensors for these molecules would help solve crucial problems such as the detection of residual pesticides on fruits and vegetables, remaining medicine in the bloodstream, tracking quorum sensing or bacterial communication, microbial degradation, and the presence of Persistent Organic Pollutant (POP). The molecules used and some of their properties are listed below:

Molecule Descriptions

Molecule Name Description
Carabyl Carabyl (CAR) Carbaryl is a man made pesticide toxic to insects (used outdoors only: agricultural crops, lawns, home gardens, anthills, outside of homes). Over 190 registered pesticide products contain carbaryl. This pesticide overstimulates the nervous system of insects that make contact with it. Normally, an enzyme breaks down the signaling chemical once it reaches its target on the nerve. However, carbaryl prevents the enzyme from working properly. This over stimulates the nerves, preventing the breathing muscles from contracting, and killing the insects. Carbaryl is also moderately toxic to humans when ingested and has low toxicity levels when inhaled. The chemical breaks down into 1-naphthol, which can be used as a biological indicator of Carbaryl. The molecular eight is 201.22 g/mol, the solubility is 40 g/L, and the ideal solvent is DMSO or ethanol. [5]
PBA

3-phenoxybenzoic acid (PBA)

 3-phenoxybenzoic acid is a metabolite of pyrethroid insecticide with low mammalian toxicity. It is a prominent environmental contaminant because its a degradation product of pyrethroid (a group of man made pesticides) insecticides. The molecule is widely used in agriculture and household pest control. As a degradation product, it can persist in environments, is very toxic to aquatic life, and can be found in soil and bodies of water where pyrethroid insecticides have been used. The molecular weight is 214.22 g/mol, the solubility is 25g/L, and the ideal solvent is DMSO. [18]
LOV Lovastatin (LOV) Lovastatin is a medication that belongs to the drug class of statins, which are used to lower cholesterol levels in blood. It is an inhibitor of HMG-CoA reductase (an enzyme related to the synthesis of cholesterol in the liver, which converts HMG-CoA to mevalonate). Lovastatin reduces production of low density lipoprotein (LDL) cholesterol. It is also used in drugs to lower cholesterol levels and helps prevent cardiovascular diseases such as heart attacks or strokes. [9] Its molecular weight is 404.54 g/mol and its solubility is 20g/mol in DMSO. [10]
BHL Butanoyl-homoserine lactone (BHL) Butanoyl-homoserine lactone is a signaling molecule used in quorum sensing (a process of bacterial communication which regulates gene expression in response to changes in cell population density). The molecule is also involved in controlling gene expression and cellular metabolism. It diffuses freely across the bacterial cell membrane and is used in synthetic biology to engineer bacterial systems with customized gene expression profiles. The molecular weight is 171.19 g/mol and the solubility is 100 g/L in the ideal solvent of DMSO. [15]
PGA Phenylglyoxylic Acid (PGA) Phenylglyoxylic Acid is an exposure biomarker used in environmental and occupational health to monitor the level of styrene, and other related compounds. This molecule helps assess exposure levels and prevent potential health risks related to these compounds. PGA itself is not highly toxic. Rather, as a breakdown product of styrene, which is used to make plastics and rubber,the presence of PGA indicates exposure to potentially harmful compounds. The molecular weight is 150.13 g/mol and the solubility is 30 g/L in the ideal solvent of DMSO. [3]
PRO Propoxur (PRO) Propoxur is a carbamate insecticide used to control a variety of pests, including insects and other arthropods. It inhibits the enzyme acetylcholinesterase, causing an accumulation of acetylcholine at nerve synapses and continuous nerve signal transmissions. These effects ultimately paralyze and kill the insects. Additionally, acute exposure of humans to propoxur can cause short term issues such as nausea, sweating, and vomiting. Long term exposure to propoxur can also cause decreased cholinesterase levels. However, other symptoms related to decreased body weight and bladder and liver issues are still being investigated. The molecular weight is 209.245 g/mol and it is soluble in the ideal solvent of ethanol. [14]
PFS Perfluorooctane sulfonate (PFS) Perfluorooctane sulfonate is a man-made fluorosurfactant and global pollutant. It was added to the Stockholm Convention on Persistent Organic Pollutant (POP) list in 2009. Recent studies show that it can cause cancer and cause developmental toxicity according to the Environmental Protection Agency (EPA) [13]. Additionally, the molecule, commonly found in synthetic chemicals used in stain repellents and fabric protectors, is found to be associated with chronic kidney disease. The molecular weight is 500.13 g/mol, solubility is 0.68 g/L, and the ideal solvent is water. [12]
CND Cis-Naphthalene Dihydrodiol (CND) Cis-Naphthalene Dihydrodiol is an intermediate in the microbial degradation of naphthalene (an insecticide, pest repellent, and common environmental pollutant). As a result, this molecule can be used as a biomarker. It is generally less toxic than napthalene but exposure to it can still be harmful. Additionally, Cis-Naphthalene Dihydrodiol can help develop effective strategies for managing naphthalene pollution. The molecular weight is 162.18 g/mol, solubility is 22.5 g/L, and ideal solvent is water. [19]
DEP

Diethyl Phthalate (DEP)

 Diethyl Phthalate is a synthetic substance used to make plastic more flexible. It is commonly used in toothbrushes, automobile parts, tools, toys, food packaging, and cosmetics. Diethyl Phthalate can also be released fairly easily from these products. The molecule is a clear, colorless liquid without significant odor so it can be easily diluted with other liquids. The primary hazard is to the environment (easily penetrates soil and contaminates groundwater). The molecular weight is 222.24 g/mol. [7]
TAR Tartaric Acid (TAR) Tartaric Acid occurs naturally in many plants, especially in grapes. This compound is a type of alpha hydroxy acid (AHA) that are natural acids found in foods. It is commonly used to generate carbon dioxide through interaction with sodium bicarbonate and acts as a muscle toxin by inhibiting the production of malic acid. Tartaric Acid, commonly used in wine to adjust acidity levels, is safe in small amounts. It is also used in cosmetics to exfoliate skin. The molecular weight is 150.087 g/mol and is highly soluble in the ideal solvent of water. [11]

Ultimately, we hope to make relevant contributions to the fundamental understanding of synthetic biology for other scientists to use in the future.

References

[1] Zaslaver, A., Bren, A., Ronen, M., Itzkovitz, S., Kikoin, I., Shavit, S., Liebermeister, W., Surette, M. G., & Alon, U. (2006). A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nature methods, 3(8), 623–628. https://doi.org/10.1038/nmeth895.
[2] Feng Y, Xie Z, Jiang X, Li Z, Shen Y, Wang B, Liu J. (2018) The Applications of Promoter-gene-Engineered Biosensor, Sensors, 18(9), https://doi.org/10.3390/s18092823.
[3] Benzoylformic acid | C8H6O3 | CID 11915. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/Benzoylformic-acid.
[4] Bhatia, D., Paul, S., Acharjee, T., & Sundar Ramachairy, S. (2023, July 23). Biosensors and their widespread impact on human health. Science Direct. Retrieved September 14, 2024, from https://www.sciencedirect.com/science/article/pii/S2666351123000311.
[5] Carbaryl | C12H11NO2 | CID 6129. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/Carbaryl.
[6] Chapman, M. J., Wallace, E. C., & Pollock, T. A. (2020). Organic Acid Profiling. In Elsevier eBooks (pp. 236-244.e6). https://doi.org/10.1016/b978-0-323-43044-9.00029-7.
[7] Diethyl Phthalate | C12H14O4 | CID 6781. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/Diethyl-Phthalate.
[8] Introduction to biosensors - PMC. (2016, June 30). NCBI. Retrieved September 14, 2024, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986445/.
[9] Lovastatin. (n.d.). MedlinePlus. Retrieved September 14, 2024, from https://medlineplus.gov/druginfo/meds/a688006.html.
[10] Lovastatin | C24H36O5 | CID 53232 - PubChem. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/Lovastatin.
[11] L-Tartaric acid | C4H6O6 | CID 444305. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/L-Tartaric-acid.
[12] Perfluorooctanesulfonic acid | C8F17SO3H | CID 74483. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/Perfluorooctanesulfonic-acid.
[13] PFOS (Perfluorooctane Sulfonate or Perfluorooctane Sulfonic Acid) - Proposition 65 Warnings Website. (n.d.). P65Warnings.ca.gov. Retrieved September 14, 2024, from https://www.p65warnings.ca.gov/fact-sheets/pfos-perfluorooctane-sulfonate-or-perfluorooctane-sulfonic-acid.
[14] Propoxur | C11H15NO3 | CID 4944. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/Propoxur.
[15] PubChem. (n.d.). N-Butyrylhomoserine lactone. PubChem. https://pubchem.ncbi.nlm.nih.gov/compound/N-Butyrylhomoserine-lactone.
[16] Siegel, M. S., & Isacoff, E. Y. (2004, January 7). Green fluorescent protein-based sensors for detecting signal transduction and monitoring ion channel function. Science Direct. Retrieved September 14, 2024, from https://www.sciencedirect.com/science/article/abs/pii/S0076687900272819.
[17] Vernhet, A. (2019). Red wine clarification and stabilization. In Elsevier eBooks (pp. 237–251). https://doi.org/10.1016/b978-0-12-814399-5.00016-5.
[18] 3-Phenoxybenzoic acid | C13H10O3 | CID 19539. (n.d.). PubChem. Retrieved September 14, 2024, from https://pubchem.ncbi.nlm.nih.gov/compound/3-Phenoxybenzoic-acid.
[19] (1R, 2S)-cis 1,2 dihydroxy-1,2-dihydronaphthalene: Uses, Interactions, Mechanism of Action | DrugBank Online. (n.d.). DrugBank. https://go.drugbank.com/drugs/DB08264.