Why Detect Triclocarban in Sludge?
Triclocarban is a common antimicrobial agent found in consumer and personal care products. It serves as a disinfectant, an antiseptic drug but most of all, an environmental contaminant. Triclocarban is also and endocrine-disrupting chemical.[1] Endocrine-disrupting chemicals (EDCs) are exogenous chemcials or chemical compositions that have the potential to significantly alter a variety of imporatnt biochemical pathways and affect the endocrine system.[2]
The average concentration of triclocarban found in the sludge of wastewater treatment plants is about 21,000 ng/g or 21 ppm on a dry weight basis (dm) in primary sludge and 13,000 ng/g or 13 ppm dm in secondary sludge.[4] Therefore, detecting triclocarban and measuring its concentration requires powerful analytical techniques.
Current Detection Methods:
Triclocarban is generally detected using High-Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), Liquid Chromatography-Mass Spectrometry (LC-MS), Solid-Phase Extraction (SPE), Nuclear Magnetic Resonance (NMR) and fluorescence detectors.[5]
Specialized sample preparation techniques for collecting and isolating triclocarban from a complex environment is required for detection through HPLC. Specialized Liquid Chromatography columns will be required for effective chromatographic separation and analysis of triclocarban. HPLC and LC-MS are also non-portable detection techniques. In the case of NMR analysis, a rich polar environment would make the spectra of NMR very complex and will complicate the process of TCC detection.
Ideation:
Our team came up with various initial ideas aiming to design a portable and highly sensitive sensor to detect the concentration of triclocarban in parts per billion. A few detection methods were explored as well as their merits and demerits which have been summarized henceforth.
Electrochemical Sensors:
Electrochemical sensors are used to detect various compounds in wastewater at parts per million levels. It consists of an electrode and a material coated transducer. A material which can have redox reactions with the analyte is chosen to coat the electrode. As the reaction takes place, the transducer connected to the electrode converts the chemical signal to an electrical signal which can then be detected.[6]
Triclocarban possesses free ions such as chlorine ion (Cl-) which can produce a signal by interacting with the positive electrode. Unfortunately we found no literature on triclocarban’s redox reactions. Hence, even if we go by the assumption that these chlorine ions will interact with the electrode, this sensor would still have low selectivity as there are many compounds possessing chlorine ions. Moreover, employing this in a bioreactor setup would not be feasible because then our sensor must undergo selective coating, the sample would have to undergo pretreatment and proper calibration has to be done.
Enzyme-based Sensors:
Enzyme-based biosensors are sensors whose recognition element is an enzyme which is immobilized on the surface of the sensor. The enzyme is chosen based on its binding affinity to the analyte. When the analyte binds to the enzyme, the enzyme undergoes some physiochemical changes which are converted to a measurable signal by the transducer present in the sensor which is then detected.[8]
An amidase from Ochrobactrum sp. TCC-2 is capable of degrading triclocarban and possesses a high binding affinity towards this compound. However, after consulting experts, we found that due to the trace levels of triclocarban in effluent, significant modifications would have to be made to the biosensor in order to make it sensitive to triclocarban. Given our time constraint, this detection method was not feasible.
Aptasensors:
Since most of our ideas were discarded due to off-target effects, we were looking for something that will specifically bind to triclocarban. This is when we came across aptamers, also known as artificial antibodies. They are single-stranded deoxyribonucleic acids (ssDNA) or ribonucleic acids (RNA), specific to a certain compound and obtained by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. These aptamers are used as detector molecules on a biosensor.[10]
Through our literature survey, we found that there are no existing aptamers for triclocarban which meant that we would either have to construct it in our lab using SELEX or employ an in-silico method of aptamer construction. SELEX is an expensive and time consuming procedure, while in-silico methods of aptamer construction require SELEX to validate the oligonucleotide library produced and then isolate a specific aptamer [11]. Due to these constraints and the lack of resources, we decided to explore further avenues.
Nanobodies-based Sensors:
Nanobodies, also known as VHHs (variable domain of heavy chain of immunoglobulin) are variable fragments of camelid-heavy chain antibodies. Due to their small size, they can be extremely selective while binding to epitomes. They provide high stability, versatility, affinity and specificity.
Three nanobodies T4, T9 and T10 were found to bind to triclocarban. We explored the option of detecting this interaction and further built upon these findings using recent detection techniques.[13]
Usually, Gold nanoparticles conjugated with single-domain antibodies are employed to enhance the detection signal. It was found that the utilization of antibody-modified gold nanoparticles offers advantages in terms of immobilizing a greater quantity of antibody onto the electrode. This is to increase the sensitivity of electrochemical impedance sensors.[14]
We also found that a selective detection limit of picograms per milliliter (pg/mL) using the llama antibody-templated gold particles for cholera toxin detection has been done. Llama VHHs were found to possess the remarkable capability of differentiating between closely related molecules with a high degree of similarity. We discovered that TCC binds to Llama single domain antibodies. Llama single domain antibodies have been subjected to sequencing, cloned into an expression vector, and expressed in Escherichia coli. They have also been generated in large quantities within Saccharomyces cerevisiae.[15] However since the purification of single domain antibodies is expensive, we decided to explore other horizons for a cheaper alternative.
Molecularly Imprinted Polymers based Sensors:
Molecularly imprinted polymers (MIPs) are synthetic polymers that mimic the structure of the compound to be detected [16]. The utilization of molecularly imprinted polymers has significantly enhanced the sensitivity of traditional chemical sensors.
We aimed to find a selective recognition polymer for triclocarban and to integrate the detection system into the wastewater treatment plant or propose it along with our bioreactor. Molecularly imprinted polymers for the detection of triclocarban and triclosan have been published. However, the polymer recognizes both of these compounds, due to which we need to create a novel polymer which is highly sensitive and selective towards TCC.
Molecularly Imprinted Solid-Phase Extraction (MISPE) was shown to significantly improve the extraction of TCC, achieving an impressive extraction rate of 89.9%. The usage of LC-MS or GC–MS without MISPE, detected around 60 times lower percentage of Triclocarban compared to those obtained using MISPE-HPLC-UV.[17]
This approach would require extensive experimental data which analyzes various interactions in the sludge of the wastewater treatment plant and experimentation needs to be done to build a polymer. Therefore, due to time constraints, we decided to look for other feasible options.
Nano-material based Sensors:
As previously stated, triclocarban is an EDC(Endocrine disrupting chemicals). Nanomaterials have shown ability to identify EDCs down to picomole or single molecule level and have been identified as viable tools for analyzing trace-level chemical species. Additionally, they can be employed as pre-treatment steps for LC-MS and GC-MS. Before doing chromatographic analysis, appropriate sample preparation techniques must be used due to the complexity of certain environmental matrices and given that EDCs are often present in low quantities.
A suitable sample preparation method allows the preconcentration of the analytes, a reduction in the matrix effect by the removal/separation of the analytes from interferers in the matrix, and the transfer of the analytes to a medium suitable for the analytical instrumentation. To achieve this, it has been seen that employing nanomaterials in sorptive extraction methods and membrane-assisted solvent extraction proves to be beneficial.[20] Electrochemical sensors and biosensors integrating nanomaterials have shown great potential to improve the sensitivity, selectivity, and response speed for EDC detection due to the particular chemical and electrical characteristics displayed by nanomaterials.[21]
We could not proceed with this idea as no nanomaterial has been isolated for the detection of triclocarban and we would have to make inferences from the nanomaterials used in triclosan detection. Moreover, there are a number of nanomaterials used in the detection of triclosan (some of which were expensive) and due to some constraints, we were unable to conduct experiments to optimize it for triclocarban.
Our Solution:
Lab-scale detection: (Hardware)
Initially, our aim was to detect triclocarban in the effluent which would require the detection of the compound in concentrations of parts per billion. During the course of our project, we decided to shift our focus to degrading triclocarban in the sludge over the effluent. Since triclocarban is present in parts per million in the sludge, a system that would detect the compound in parts per million would be sufficient.
After analyzing and learning from numerous approaches, as listed above, we decided to simplify our approach, and landed on a detection system which can be integrated with our bioreactor. This consists of a laser, photodiode and infrared filter placed inside a sample collector. The initial and final samples of feed would be taken out manually from the inlet and outlet pipe of the bioreactor via valves. The sample would then be placed into the sample collector, where monochromatic light from the laser would interact with the compound. If the presence of photons within a specific wavelength range are identified, the photodiode coupled with an infra-red (IR) filter would capture these photons and confirm the presence of triclocarban in the given sample. Typically multiple wavelength ranges are isolated for a particular compound using Raman Spectroscopy, and these wavelength ranges can be integrated to design a photodiode particular to the compound. By attaching a microcontroller to the photodiode, data can be wirelessly transmitted to a software, where reduction of background noise and quantification of triclocarban would be done.
Raman Spectroscopy was used in order to identify specific bond vibrations (peaks) in the structure of triclocarban. Raman spectroscopy, unlike other elemental analysis, is a non destructive method and does not change the samples composition or structure and offers higher resolution compared to other spectral methods.[23][24]
Using an excitation laser of wavelength 785 nm, Surface Enhanced Raman Spectral analysis of triclocarban was analyzed. The Raman Spectra for 1 part per million and 100 parts per million triclocarban gives a unique peak at 758.34644 cm-1. In the 10 parts per million sample, a Raman shift is seen towards the left, and the peak is detected at 754.38551cm-1. Using this data, we can come to a conclusion that the Raman Shift should be around 750-760 cm-1 (wavenumber).
The wavelength that detects this Raman shift was calculated using the formula:
The wavelength was identified as = 834.9185474 nm. In order to acquire the specific wavelength, we plan on using an IR filter of wavelength range 820 to 840 nm. Additionally, A silicon PIN photodiode within the range 350 to 1100 nm was selected. A microcontroller attached to this photodiode would wirelessly transmit the data to a software which would be programmed to quantify triclocarban using the standard calibration curve.
Lab-scale detection: (Wet Lab- Future Implementation)
To quantify the TCC degradation efficiency of TccA amidase, we need to perform expression studies. To detect the loss in TCC, we have come up with a method that includes a Thin Film Solid Phase MicroExtraction (TFSPME). This membrane can adsorb TCC and other non polar compounds. This is done by vigorously moving the membrane in the broth and then eluting the TCC with Acetonitrile (HPLC grade).
We plan on extracting TCC from the broth containing E.coli Bl21 before inoculation and 2 days after inoculation to check for the efficiency of the enzyme. We also plan on taking multiple readings in the middle of our 2 day span, to get the kinetics of the enzyme based on the concentration of TCC.
These samples along with the standard concentrations of TCC will be sent to LCMS or GCMS to get the concentrations of our samples. We also plan on sending standard solutions for 4 Chloroaniline and 3,4-Dichloroaniline, to check for the rate of production of by-products. UV-VIS Detection is not possible as the by-products formed have a very similar Lambda max to TCC.
References
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