Material and method

4.1 Chemicals and Materials

All the reagents used were of analytical grade. Poly acrylamide hydrochloride (PAHCl) was obtained from Otto Chemie Pvt. Ltd., PSS was obtained from Thermo Fisher Scientific, KMnO4 was obtained from Sisco research laboratories Pvt. Ltd., TMAO, and TMB were from TCI, Japan, IACN from Alfa-Aeser, trimethylamine was obtained from Spectrochem Pvt. Ltd. NH4OH was obtained from Finar Limited; Tris-HCl and NaCl were obtained from Thermo Fisher Scientific. Luria-Bertani Medium was obtained from HiMedia, and the DNA ladder and protein Marker were from Banglore-GeNei.
Preparation of reagents and stock solutions: TMB stock solution was prepared fresh in 50% glacial acetic acid. IACN solution was always prepared fresh in ethanol; these solutions were kept in amber tubes and stored in a cold cabinet. In deionized water, stock solutions of NH4OH (100 mM), HCl (5 M), TMAO (100 mM), and Tris-HCl buffer of pH 7.2 were prepared. A stock solution of PAHCl (1g/L) and PSS (1g/L) was prepared in 0.1 M NaCl.

4.2 Instruments

Spectrophotometric analysis was performed on EvolutionTM 300 UV-Vis spectrophotometer (Thermo Fisher Scientific), while Zeta and DLS were carried out on the Malvern Zetasizer (Malvern). Scanning electron microscopy was performed on the FEI Nova SEM 450.

4.3 PAHCl@MnO2nanoparticle synthesis and characterization

PAHCl@MnO2 nanoparticles were synthesized as described by (Chang et al. , 2021b) with certain modifications. To KMnO4, PAHCl solution was added and stirred vigorously at 800 rpm for 20 minutes; a colour change was observed from dark pink to brown (inset, Fig. S2(a), colour change from left to the one on the right). The formation of MnO2 was confirmed using UV absorbance [Fig. S2(a)]. These nanoparticles were added to the PSS solution dropwise while stirring and then left for 15 minutes. The solution was centrifuged at 12000 rpm for 15 minutes to remove unreacted starting materials, resuspended in PAHCl solution, and stirred for 30 minutes, followed by washing three times with water. The capping can be monitored by measuring each layer’s zeta potential change [Fig. S2(b)]. Layer 1 shows PAHCl capping, layer 2 shows PSS caping and layer 3 is PAHCl. Hence, the final product is PAHCl@MnO2nanoparticles. The SEM images show that the MnO2nanoparticles are spherical [Fig. S2(c)].

Isolation and overexpression of the Tmm

Tmm gene was amplified from the bacterial strain,Paracoccus sp . DMF (Swaroop, Sughosh, & Ramanathan, 2009). The whole genome sequence of Paracoccus sp. DMF has been deposited at DDBJ/ENA/GenBank under the accession number SOKV00000000.2, BioProject number PRJNA528176 and BioSample number SAMN11175380. The gene was amplified using the PCR program described in Table ST1.
In a PCR reaction volume of 100µl in which 10µl buffer (10 X 15 mM MgCl2 buffer), 4 µl template (genomic DNA), 10 µl forward primer (100 µM), 10 µl reverse primer (100 µM), 10 µl dNTPs (10 mM), 0.8 µl Taq Polymerase (5 U/µl) and remaining nuclease-free water was used.
The amplified gene was cloned in a pet22b (+) vector within NdeI and Hind III restriction sites with ampicillin-resistant markers. The positive clone was transformed into BL21(DE3) competent cell, spread on LB agar (1.8%) + ampicillin (50 µg/ml) plate, and incubated at 37 °C overnight. A single colony was inoculated into LB broth containing ampicillin and incubated overnight at 37 °C. A 1% v/v inoculum was taken from the previous and inoculated fresh 10 ml LB media. When the O.D. reached 0.6 - 0.65, 1 ml of culture was aliquoted as uninduced, and the rest of the culture was induced with 0.5 mM IPTG and incubated at 37 °C for 4 hours. Further, the uninduced and induced fractions were checked for over-expression on 12% SDS gel.

Cell Growth

The cells were grown by inoculating 1% v/v culture to terrific broth media + ampicillin (50 µg/ml) and incubated at 37 °C till OD reached 0.7 - 0.8, after which the culture was induced with 0.5 mM IPTG and incubated at 16°C for 18 hours. The bacterial cells were harvested at 4000*g, and pellets were stored at -20 °C till further use.

4.6 TMAO Assay Optimization

pH optimization : The detection of TMAO using TMB is possible in an acidic medium. The reaction is carried out at different pH ranges, varying from 1-3, to obtain an optimum range for the detection.
The derivatization of trimethylamine : To remove trimethylamine from the reaction mixture, iodoacetonitrile (IACN) was used. (Hefniet al. , 2021) A slightly excess amount of IACN and ammonium hydroxide was constantly added to the analyte solution and incubated at room temperature for 15 minutes. After the trimethylamine derivatization, TMAO analysis was carried out.
Scheme 2: Reaction of IACN with trimethylamine to form the derivative 3.
Trimethylamine derivatization using IACN was confirmed through ESI-MS. After incubating it with IACN for 15-20 minutes, the reaction detected no trimethylamine. (Figure S3)
TMAO detection protocol : Colorimetric detection of TMAO was performed by adding the NH4OH solution (5 mM) to a reaction buffer containing trimethylamine, then IACN solution, and incubation for 15 minutes. After the incubation period, 10 mM HCl was added, followed by 8 µL PAHCl@MnO2, five mM TMB, and another incubation for 3 minutes before taking UV of the sample. TMAO was added to the reaction, and a change in the UV spectra was observed.