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.