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Detection of the pathogenic α-proteobacterium Ochrobactrum anthropi via magnetic
enrichment and in-situ PCR using pH responsive BSA@Fe3O4 nanoparticles prior to
electrophoretic separation
Arpita P. Tiwaria, Sonali S. Rohiwala, Mangesh V.Suryavanshib, Saral J. Ghosha, Shivaji H.
Pawara*
a
b
Center for Interdisciplinary Research, D. Y. Patil University, Kolhapur 416006, MS,India
National centre for cell sciences (NCCS), Pune, 411021, MS, India
*Corresponding author, E-mail: [email protected]
Supplementary 1
Figure S1 Effect of pH on (a) adsorption (b) desorption of calf thymus DNA onto Fe3O4@BSA
magnetic nanoparticles (c) Adsorption isotherm of DNA on the Fe3O4@BSA magnetic
nanoparticles
Supplementary 2
The Langmuir isotherm theory assumes monolayer coverage of adsorbate over a
homogeneous adsorbent surface, i.e. the surface consists of identical sites, equally available for
adsorption and with equal energies of adsorption. Therefore, at equilibrium, a saturation point is
reached where no further adsorption can occur. Adsorption is assumed to take place at specific
homogeneous sites with the adsorbent and once DNA molecule occupies a site, no further
adsorption can take place at the site.. The qm is the surface concentration at monolayer coverage
in mg g-1 and illustrates the maximum value of qe that can be attained as Ce increases. The b
parameter is a coefficient related to the energy of adsorption and increases with increasing
strength of the adsorption bond. The Langmuir constants, qm and b, can be determined from the
linear plot of Ce/qe versus Ce, which has a slope of 1/qm and an intercept of 1/b qm. The linear
form of Langmuir equation is given by:
The experimental DNA uptake values have also been analyzed by using Freundlich equation. In
the experimental DNA uptake values have also been analyzed by using Freundlich equation. In
the Freundlich adsorption isotherm, the model assumes that the adsorbent consists of a
heterogeneous surface composed of different classes of adsorption sites. The Freundlich
constants, n and KF, are obtained from the linear regression analysis of the following equation:
where qe is the maximum uptake capacity and Ce is the equilibrium concentration. Plot of log qe
versus log Ce should give a straight line with a slope of 1/n and an intercept of log K F. The
parameters KF and n are the constants from the Freundlich equation representing the capacity of
the adsorbent for the adsorbate and the reaction order, respectively.
Langmuir model
Freundlich model
qmax
588 µg/mg
KF
6.456
b
10.60
n
1.49
R2
0.992
R2
0.978
Supplementary 3
a
b
Figure S2 Effect of temperature on percentage DNA adsorption.
This nature can be explained theoretically by using the following equation
N=N0·e EF/KT
(1)
where N= concentration of DNA adsorbed on magnetic core-shells at temperature, T (T=
20,40,60,80oC), N0= initial concentration of DNA present in the solution, E F= formation energy
required for adsorption of DNA, k= Boltzmann’s constant and T= temperature in oC.
Further, this equation can be modified as,
(2)
From the above equation, N/No indicates adsorption of DNA on Fe3O4@BSA magnetic
nanoparticles and is inversely proportional to the temperature, showing that as temperature
increases there is an increase in adsorption of DNA on Fe3O4@BSA magnetic nanoparticles.
Negative sign indicates that heat is used (i.e. endothermic) in the adsorption process. According
to equation the log of percentage adsorption of DNA on Fe3O4@BSA magnetic
nanoparticlesverses1/T is plotted and shown in Fig. Linear nature of the plot shows that the
adsorption process obeys equation.
Supplementary 4
Original DNA sequence
>gi|602750529|gb|KF864675.1| Ochrobactrum anthropi OX_B 16S ribosomal RNA gene, partial
sequence
TGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGCCCC
GCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCATTTGCTAC
GGAACAACAGTTGGAAACGACTGCTAATACCGTATGTGCCCTTCGGGGGAAAG
ATTTATCGGCAAATGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAG
GCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTG
GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGG
ACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAG
GGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGC
CCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGT
TCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGACTTTTAAGTCAGGGGTGA
AATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGGAAGTCTTGAGTATG
GTAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAG
GAACACCAGTGGCGAAGGCGGCTCACTGGACCATTACTGACGCTGAGGTGCGA
AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGA
TGAATGTTAGCCGTCGGGGAGTTTACTCTTCGGTGGCGCAGCTAACGCATTAA
ACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGG
GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAAC
CTTACCAGCCCTTGACATACCGGTCGCGGACACAGAGATGTGTCTTTCAGTTCG
GCTGGACCGGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATG
TTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTA
GTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGAT
GACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGG
TGGTGACAGTGGGCAGCGAGCGTGCGAGCGCAAGCTAATCTCCAAAAGCCATC
TCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAA
TCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCC
CGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGCTGTGCTAACCGCAAGGA
GGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAG
CCGTAGGGGAACCTGCG
Supplementary 5
Fig S3. Magnetic enrichment of DNA with respect to time.
The experiment was performed to concentrate the total DNA present in 5ml sample to 10 µL of
sample by varying time over magnetic field. It has been found that all 54mg of DNA has been
successfully concentrated to 10 µL volume, hence causing 150th fold increase in DNA
concentration.