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ArticlePDF Available


BACTERIA CAPTURE, LYSATE CLEARANCE, AND PLASMID DNA EXTRACTION USING
PH-SENSITIVE MULTIFUNCTIONAL MAGNETIC NANOPARTICLES

 * November 2009
 * Analytical Biochemistry 398(1):120-2

DOI:10.1016/j.ab.2009.11.006
 * Source
 * PubMed

Authors:
Zhi Shan
 * Sichuan Agricultural University



Qi Wu
 * Sichuan Agricultural University



Xianxiang Wang
 * East China University of Technology



Zhongwu Zhou


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Citations (48)
References (17)
Figures (2)





ABSTRACT AND FIGURES

A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was
developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the
pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the
subsequent removal of genomic DNA/protein complex after lysis can be achieved
simply by magnetic separation. Furthermore, the yield and purity of pDNA
extracted by MNPs are comparable to those obtained using organic solvents or
commercial kits. This time- and cost-effective protocol does not require
centrifugation or precipitation steps and has the potential for automated DNA
extraction, especially within miniaturized lab chip applications.
Cell capture efficiency as a function of fermentation culture pH. (A) Cell
densities (OD 600 ) ranged between 0.07 and 1.0, whereas the separation time and
… 
Cell capture efficiency as a function of fermentation culture pH. (A) Cell
densities (OD 600 ) ranged between 0.07 and 1.0, whereas the separation time and
MNP volume were 10 min and 10 ll, respectively. (B) MNP volumes ranged between
10 and 75 ll, whereas the separation time and cell density (OD 600 ) were
… 



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Notes & Tips
Bacteria capture, lysate clearance, and plasmid DNA extraction using
pH-sensitive multifunctional magnetic nanoparticles
Zhi Shan
a
,QiWu
a
, Xianxiang Wang
a
, Zhongwu Zhou
a
, Ken D. Oakes
b
, Xu Zhang
b
, Qianming Huang
a
,
Wanshen Yang
a,*
a
Faculty of Science, Sichuan Agricultural University, Yaan 625014, PR China
b
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
article info
Article history:
Received 30 July 2009
Received in revised form 17 October 2009
Accepted 3 November 2009
Available online 10 November 2009
abstract
A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was
developed to isolate
plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive
carboxyl-modified magnetic
nanoparticles, both cell capture and the subsequent removal of genomic
DNA/protein complex after lysis
can be achieved simply by magnetic separation. Furthermore, the yield and purity
of pDNA extracted by
MNPs are comparable to those obtained using organic solvents or commercial kits.
This time- and cost-
effective protocol does not require centrifugation or precipitation steps and
has the potential for auto-
mated DNA extraction, especially within miniaturized lab chip applications.
Ó2009 Elsevier Inc. All rights reserved.
Because plasmid DNA (pDNA)
1
is routinely used as a genetic
engineering vector, the development of a rapid, simple, and cost-
effective pDNA extraction method is of considerable advantage. Con-
ventional pDNA extraction techniques (requiring centrifugation, pre-
cipitation, and chromatography separation) are not easily adapted to
automated systems. In contrast, magnetic nanoparticle extraction
methods demonstrate remarkable simplicity owing to the nature
of the particles, which serve as a DNA adsorbent within biological
matrices [1,2]. To date, an array of surface-modified magnetic nano-
particles (MNPs), both chemically and biologically synthesized [1–9],
have been successfully used for pDNA purification. Various proce-
dures have been developed using MNPs with carboxyl [2–4], hydro-
xyl [5,6], and amino functional groups [7–9]; however, the
underlying mechanism is the same, namely, adsorbing pDNA from
cleared lysate [4–9]. Most MNP procedures involve two centrifuga-
tion steps: the first to harvest cells from liquid culture and the sec-
ond to pellet denatured genomic DNA/protein complexes after cell
disruption and neutralization. These centrifugation procedures are
both time- and labor-intensive; moreover, this step was not amena-
ble to the miniaturization and automation required of high-through-
put biological sample preparation [2]. Furthermore, the potential for
shearing damage to biomacromolecules during extensive centrifuga-
tion is unavoidable.
The current study demonstrated a centrifugation-free proce-
dure for pDNA extraction from Escherichia coli using carboxyl-mod-
ified MNPs. This approach used magnet-assisted separation to
harvest both E. coli cells from a fermentation culture and denatured
genomic DNA/protein complexes after lysis. The quality and quan-
tity of magnetic particle-purified pDNA were confirmed by com-
parison against pDNA obtained using organic solvents and a
commercially available purification kit.
Carboxyl-modified superparamagnetic nanoparticles were used
as a multifunctional bioadsorbent [10]. FeCl
3
and FeSO
4
(in a molar
ratio of 1.65 in a 4-M NaOH solution) were used to prepare Fe
3
O
4
MNPs [4]. The Fe
3
O
4
MNPs were later separated and further coated
by polymerization of monomer methacrylic acid using an emulsion
polymerization approach [10]. The coated MNPs were washed five
times with deionized water to remove residual methacrylic acid
monomers and other impurities. Then MNPs were dispersed in
deionized water under sonication for 10 min to form a stable mag-
netic nanofluid (17 mg/ml by dry weight) that can be stored for
several months at room temperature.
The cell capture efficiency (CCE) of MNPs was evaluated using
E. coli JM109 containing pET 15b vector as a model organism. Cells
were grown in Luria–Bertani medium (pH 7.0) containing 50
l
g/ml
ampicillin at 37 °C overnight. For optimization of cell capture, two
experiments were performed. The first investigated the effect of pH
on CCE. In this experiment, 10
l
l of MNPs was added to 1.5 ml of
differing E. coli concentrations (determined by optical density
[OD] at 600 nm), with OD values ranging from 0.07 to 1.0 using
cell-free culture supernatant as diluent. Each cell concentration
was pH adjusted (using 1 M HCl) to achieve a range of pH values
(1.18–7.0). Then Eppendorf tubes containing the cells (varying in
0003-2697/$ - see front matter Ó2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2009.11.006
*Corresponding author. Fax: +86 835 2886136.
E-mail address: wansheny@163.com (W. Yang).
1
Abbreviations used: pDNA, plasmid DNA; MNP, magnetic nanoparticle; CCE, cell
capture efficiency; OD, optical density; SDS, sodium dodecyl sulfate; EDTA,
ethyl-
enediaminetetraacetic acid; PEG, polyethylene glycol; UV, ultraviolet.
Analytical Biochemistry 398 (2010) 120–122
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio


















pH and concentration) were placed in a magnetic field generated
by a Promega magnetic separation stand for 10 min [4]. After
immobilization of the cell/nanoparticle complexes, the superna-
tant was assessed for optical density at 600 nm. The second exper-
iment addressed the influence of MNP concentration on CCE,
where the E. coli cell concentration was held constant (OD
600
of
1.0, 1.5 ml). In this experiment, the magnetic separation time
was set at 3 min, and adjusted pH values again ranged between
1.18 and 7.0 while the volume of MNP fluid added to each cell ali-
quot ranged between 10 and 75
l
l. The CCE was calculated in
terms of the difference in optical density before and after separa-
tion: CCE (%) = 100(ab)/a, where aand brepresent the OD
600
readings before and after magnetic separation, respectively.
The results indicated that capture of cells with MNPs was essen-
tially a pH-dependent process (Fig. 1). Less than 10% of total E. coli
cells were captured between pH 5.0 and 7.0. However, when the
pH was below 5.0, CCE increased sharply, achieving upward of
90% capture between pH 2.3 and 3.2 (Fig. 1A). E. coli bacteria cell
walls possess a negative charge under neutral conditions owing
to abundant carboxyl, phosphoryl, and hydroxyl groups present
in macromolecules of the cell wall [11–13]. When these functional
groups are protonated with acid (or negative charges are neutral-
ized with cationic polymers), the cells readily aggregate within
the culture medium, as described previously [12–14]. Hence, the
addition of HCl will strongly suppress dissociation of these ionic
groups on the cell wall [12–14] and carboxyl groups on the MNP
surface, resulting in colloidal instability and flocculation of both
E. coli cells and MNPs. The resultant cell/MNP complexes present
within low-pH environments contributed to the increased CCE.
However, a slight CCE decrease was observed below pH 2.0
(Fig. 1A), possibly due to partial restabilization of E. coli cells as a
result of amino group ionization on the cell surface.
Concentrations of cells and MNPs also played an important role
in CCE. For instance, at pH 3.8 the highest capture efficiency
(96.3%) was observed at an OD
600
of 1.0, whereas at lower cell den-
sities (OD
600
of 0.07) the CCE dropped to 62.1% (Fig. 1A). Further-
more, a slight increase in CCE was observed with higher MNP
concentrations (Fig. 1B), suggesting coflocculation as the bacteria
capture mechanism given that high concentrations of cells and/or
MNPs increased CCE. The main advantage of using more MNPs
(35–75
l
l) for capture was a significant reduction in separation
time. Only the lowest MNP addition (10
l
l) failed to achieve cap-
ture efficiencies greater than 90% (within appropriate pH ranges
of 2.0–3.2) with only 3 min of magnetic separation (Fig. 1B). For
subsequent studies, optimized conditions (50
l
l of MNPs, pH 3.2)
were used for cell capture. Typically, more than 95% of E. coli cells
could be recovered within 1.5 min from 1.5 ml of overnight culture
with OD
600
higher than 0.2. The separated cell/MNP complexes
could then be easily lysed for pDNA extraction. The ease of lysis
can be ascribed to the nonuniform cell aggregation exhibited by
the MNPs (Fig. 2A), that, rather than uniformly covering the entire
cell surface, leave E. coli membranes susceptible to sodium dodecyl
sulfate (SDS) and alkaline solution.
Cell lysis and pDNA purification details were as follows. Once
the cell/MNP complexes were firmly immobilized on the tube wall,
the culture supernatant was discarded. Captured cell/MNP com-
plexes were resuspended in 100
l
l of solution I (25 mM Tris [pH
8.0], 10 mM ethylenediaminetetraacetic acid [EDTA], and 400
l
g/
ml RNase A), followed by the addition of 200
l
l of solution II
(0.2 M NaOH and 1% [w/v] SDS). The resultant mixture was incu-
bated on ice for 3 min after gentle mixing. Genomic DNA, proteins,
and other cell debris were precipitated with the addition of 150
l
l
of solution III (3 M potassium acetate, pH 5.5). The precipitate with
trapping MNPs inside was separated magnetically. The cleared
alkaline lysate supernatant was transferred to a new 1.5-ml Eppen-
dorf tube for pDNA extraction. A 1/10 volume of MNPs was added
to the tube and well mixed with the supernatant by five pipetting
cycles, followed by mixing with an equal volume of binding buffer
(15% PEG [polyethylene glycol] 8000 and 2.5 M NaCl) [4]. The
MNPs were immobilized, and the supernatant was removed using
a pipet. The pellet was rinsed with 750
l
l of cold 70% ethanol. After
removal and evaporation of the ethanol, the pDNA was eluted in
50
l
l of TE buffer (10 mM Tris [pH 8.0] and 1 mM EDTA) at room
temperature for 0.5 min. The MNPs were then immobilized with
the supernatant transferred to a DNase/RNase-free Eppendorf tube.
To compare the MNP technique against existing methods, pDNA
extraction was also performed using organic solvents and a com-
mercial kit (Qiagen, USA) according to the molecular cloning labo-
ratory manual [15] and the manufacturer’s instructions,
respectively.
The yield and purity of pDNA were analyzed by ultraviolet (UV)
spectroscopy and agarose gel electrophoresis. The bands were
visualized under UV light by GoldView staining (SBS Genetech,
China) using the Gel Doc XR System (Bio-Rad, USA). In an evalua-
tion of extraction methods (performed in triplicate), comparable
pDNA was extracted by MNPs (8.57 ± 0.13
l
g) from 1.5 ml of
overnight culture as was extracted with organic solvents
(10.36 ± 0.11
l
g) and by the commercial kit (9.32 ± 0.05
l
g), as
illustrated in Fig. 2B. The pDNA purity, as estimated by the
Fig. 1. Cell capture efficiency as a function of fermentation culture pH. (A)
Cell
densities (OD
600
) ranged between 0.07 and 1.0, whereas the separation time and
MNP volume were 10 min and 10
l
l, respectively. (B) MNP volumes ranged
between 10 and 75
l
l, whereas the separation time and cell density (OD
600
) were
3 min and 1.0, respectively.
Notes & Tips / Anal. Biochem. 398 (2010) 120–122 121
















OD
260
/OD
280
ratio, was approximately 1.8, indicating that the ex-
tracted DNA was pure with negligible protein contamination. The
compatibility of the pDNA isolated by the current method was
demonstrated by a successful double restriction digestion
(Fig. 2B) with BglII and EcoRI (Sangon, China), indicating the feasi-
bility of the purified DNA for downstream applications. Because
the recovery of cells, the removal of floc after neutralization, and
the purification of pDNA all were accomplished with rapid mag-
netic separation rather than centrifugal processes, the entire proce-
dure required less than 20 min, whereas the comparable
established methods took at least 30 min. Furthermore, the current
method required less handling and no hazardous reagents such as
phenol and chloroform. The setup costs associated with the current
method are much less than those of centrifugation-dependent
methods. In addition, the E. coli cells can be captured by carboxyl-
ated MNPs rather than expensive immunomagnetic particles.
In summary, multifunctional MNPs proved to be a time- and
cost-effective pDNA preparation technique independent of centri-
fugation and hazardous organic solvents. Not only are the pH-sen-
sitive magnetic nanoparticles well suited for routine laboratory
use, but also the simplicity of this approach indicates their poten-
tial for automated pDNA purification.
Acknowledgments
This work was supported by a research grant from Sichuan
Agricultural University. The authors thank Yi Zhou for transmission
electron microscope (TEM) imaging and helpful discussions.
References
[1] Z.M. Saiyed, C. Bochiwal, H. Gorasia, S.D. Telang, C.N. Ramchand,
Application of
magnetic particles (Fe
3
O
4
) for isolation of genomic DNA from mammalian
cells, Anal. Biochem. 356 (2006) 306–308.
[2] X. Xie, X. Nie, B. Yu, X. Zhang, Rapid enrichment of leucocytes and genomic
DNA from blood based on bifunctional core–shell magnetic nanoparticles, J.
Magn. Magn. Mater. 311 (2007) 416–420.
[3] T.R. Sarkar, J. Irudayaraj, Carboxyl-coated magnetic nanoparticles for mRNA
isolation and extraction of supercoiled plasmid DNA, Anal. Biochem. 379
(2008) 130–132.
[4] Z. Shan, W. Yang, X. Zhang, Q. Huang, H. Ye, Preparation and
characterization of
carboxyl-group functionalized superparamagnetic nanoparticles and the
potential for bio-applications, J. Braz. Chem. Soc. 18 (2007) 1329–1335.
[5] M.J. Davies, J.I. Taylor, N. Sachsinger, L.J. Bruce, Isolation of plasmid
DNA using
magnetite as a solid phase adsorbent, Anal. Biochem. 262 (1998) 92–94.
[6] C.L. Chiang, C.S. Sung, C.Y. Chen, Application of silica-magnetite
nanocomposites to the isolation of ultrapure plasmid DNA from bacterial
cells, J. Magn. Magn. Mater. 305 (2006) 483–490.
[7] C.L. Chiang, C.S. Sung, T.F. Wu, C.Y. Chen, C.Y. Hsu, Application of
superparamagnetic nanoparticles in purification of plasmid DNA from
bacterial cells, J. Chromatogr. B 822 (2005) 54–60.
[8] X. He, H. Huo, K. Wang, W. Tan, P. Gong, J. Ge, Plasmid DNA isolation using
amino-silica coated magnetic nanoparticles (ASMNPs), Talanta 73 (2007) 764–
769.
[9] B. Yoza, A. Arakaki, T. Matsunaga, DNA extraction using bacterial magnetic
particles modified with hyperbranched polyamidoamine dendrimer, J.
Biotechnol. 101 (2003) 219–228.
[10] S. Yu, G.M. Chow, Carboxyl group (–CO
2
H) functionalized ferrimagnetic iron
oxide nanoparticles for potential bio-applications, J. Mater. Chem. 14 (2004)
2781–2786.
[11] M. Torimura, S. Ito, K. Kano, T. Ikeda, Y. Esaka, T. Ueda, Surface
characterization
and on-line activity measurements of microorganisms by capillary zone
electrophoresis, J. Chromatogr. B 721 (1999) 31–37.
[12] S. Barany, A. Szepesszentgyörgyi, Flocculation of cellular suspensions by
polyelectrolytes, Adv. Colloid Interface Sci. 111 (2004) 117–129.
[13] K.E. Eboigbodin, J.J. Ojeda, C.A. Biggs, Investigating the surface
properties of
Escherichia coli under glucose controlled conditions and its effect on
aggregation, Langmuir 23 (2007) 6691–6697.
[14] S.P. Strand, M.S. Vandvik, K.M. Vrum, K. Stgaard, Screening of chitosans
and
conditions for bacterial flocculation, Biomacromolecules 2 (2001) 126–133.
[15] J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, third
ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.
Fig. 2. (A) Transmission electron microscope (TEM) image of E. coli cells
captured
with MNPs. The scale bar represents 250 nm. (B) Agarose gel electrophoresis of
pDNA digested with two restriction endonucleases (BglII and EcoRI). Lanes 1, 2,
and
3: pDNA extracted with standard organic solvents, a commercial Qiagen kit, and
MNPs, respectively; lanes 4, 5, and 6: restriction digestion of pDNA extracted
with
standard organic solvents, the Qiagen kit, and MNPs, respectively; lane M: DNA
molecular weight markers (fragment sizes 0.5, 1.0, 2.0, 3.5, 5.5, and 7 kb).
122 Notes & Tips / Anal. Biochem. 398 (2010) 120–122




CITATIONS (48)


REFERENCES (17)




... However, the results for the conventional recommended culture-based
technique can take 4-7 days to be conclusive [8,9]. Numerous efforts have been
made to increase bacterial concentration as a result, including those involving
biofilms [10], centrifugation [11], dielectrophoresis [12], filtration [13], and
magnetic nanoparticles (MNPs) [14][15][16]. These more recent methods have
reduced the time from days to hours. ...

Multi-Probe Nano-Genomic Biosensor to Detect S. aureus from
Magnetically-Extracted Food Samples
Article
Full-text available
 * Jun 2023

 * Chelsie Boodoo
 * Emma Dester
 * Jeswin David
 * Evangelyn C. Alocilja

One of the most prevalent causes of foodborne illnesses worldwide is
staphylococcal food poisoning. This study aimed to provide a robust method to
extract the bacteria Staphylococcus aureus from food samples using glycan-coated
magnetic nanoparticles (MNPs). Then, a cost-effective multi-probe genomic
biosensor was designed to detect the nuc gene of S. aureus rapidly in different
food matrices. This biosensor utilized gold nanoparticles and two DNA
oligonucleotide probes combined to produce a plasmonic/colorimetric response to
inform users if the sample was positive for S. aureus. In addition, the
specificity and sensitivity of the biosensor were determined. For the
specificity trials, the S. aureus biosensor was compared with the extracted DNA
of Escherichia coli, Salmonella enterica serovar Enteritidis (SE), and Bacillus
cereus. The sensitivity tests showed that the biosensor could detect as low as
2.5 ng/µL of the target DNA with a linear range of up to 20 ng/µL of DNA. With
further research, this simple and cost-effective biosensor can rapidly identify
foodborne pathogens from large-volume samples.
View
Show abstract
... Magnetic nanoparticles (MNPs) have a large surface area due to their small
size, which allows them to be easily functionalized with different molecules
[27]. MNPs are used for the purification of various biomolecules such as DNA
[28], histidine-tagged (his-tag) protein, enzyme [29][30][31], cell [32], and
RNA [33]. Although many researchers focus on producing MNPs to purify his-tag
proteins, there are few studies on MNPs specifically functionalized for a single
enzyme [34][35][36][37][38][39]. ...

Sulfanilamide Modified Magnetic Nanoparticles for Purification of Carbonic
Anhydrase from Bovine Blood
Article
Full-text available
 * Jun 2022
 * APPL BIOCHEM BIOTECH

 * Safinur Çelik
 * Kübra Solak
 * Ahmet Mavi

Magnetic nanoparticles (MNPs) have been used for purification of specific
biomolecules form mixtures. The aim of this study is to develop a new, cheap,
reusable, and magnetic-based material to purify the carbonic anhydrase (CA)
enzyme in a short time with high efficiency. In the first part of this study,
silica-coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) were obtained.
Surface modification of Fe3O4@SiO2 MNPs was accomplished with
3-(4-Hydroxyphenyl) propionic acid (PA) and sulfanilamide (SA), respectively. SA
is a selective inhibitor of CA, and it selectively binds to CA. The final
particle was named Fe3O4@SiO2-PA-SA MNPs and characterized by SEM, TEM, XRD, and
FT-IR. It was determined that the produced MNPs contained multicore, were
smaller than 100 nm in size, and had a spherical morphology. The CA was purified
from bovine blood hemolysate in a short time such as 2.5 h and in a simple
manner. The maximum enzyme purifying capacity of MNPs was calculated as 13.87 ±
3.27 mg CA/g MNP. SDS-PAGE analysis was confirmed that high CA purification
success was achieved.
View
Show abstract
... Since most of the small plasmids do not fuse with the chromosome or are
sufficiently present in the cytoplasm, they are easy to remove from cells. For
this reason, gene transfers are generally more beneficial than small or medium
sized artificial plasmids 8 . The high number of copies in the cell is also
considered an advantage in terms of expression. ...

Plasmid DNA Isolation and Characterization Studies
Article
 * Sep 2021

 * Murat Doğan

View
... Owing to an outer membrane filled with teichoic acids of gram-positive
bacteria or lipopolysaccharides of gram-negative bacteria, the negatively
charged bacterial surface exhibits a stable negative zeta potential value with a
pH varying from 4.0 to 9.0 [24,25]. Thus, a nonselective EI strategy is
conventionally utilized in bacteria capture [26][27][28][29]. In this respect, a
polyamidoamine (PAMAM) dendrimer [30][31][32][33][34] and polyethyleneimine
(PEI) [35,36] were engineered on the surfaces of MNPs to provide highly positive
surface charges. ...

A single-tube sample preparation method based on a dual-electrostatic
interaction strategy for molecular diagnosis of gram-negative bacteria
Article
Full-text available
 * Sep 2020
 * MICROCHIM ACTA

 * Fei-xiong Chen
 * Soyeon Kim
 * Jun-Hee Na
 * Tae Yoon Lee

A single-tube method based on a dual-electrostatic interaction (EI) strategy for
bacteria capture and DNA extraction was designed to enable the highly sensitive
detection of nucleic acids. Specially designed magnetic nanoparticles were
developed to meet the opposing requirements of a single-tube method, which exist
between the strong EI required for efficient bacteria capture and the weak EI
required for DNA extraction with minimal DNA adsorption. A dual-EI strategy for
the single-tube (DESIGN) method was thus developed to integrate bacteria
enrichment, bacteria cell lysis, and DNA recovery in a single tube, thereby
minimizing precious sample loss and reducing handling time. Subsequently, we
evaluated the performance with a variety of concentrations from 5 to 100
colony-forming units (CFU)/10 mL human urine and milk samples. The DESIGN method
achieved the simple and sensitive detection of Salmonella enterica serovar
Typhimurium in 10 mL of human urine and milk samples up to 5 CFU by quantitative
PCR. Furthermore, the DESIGN method detected Brucella ovis and Escherichia coli
from 10 mL of human urine with a detection limit up to 5 CFU/10 mL. Graphical
abstract
View
Show abstract
... These particles are movable with a magnetic field and are easily removable
from the reaction medium (Pieters and Bardeletti et al. 1992). Further uses of
magnetic particles include gene therapy, thermotherapy, diagnosis, organ and
tissue targeting, drug-delivery systems, purification of enzyme and protein,
magnetic resonance imaging (MRI), and immunoassay studies (Urbina et al.
2008;Yong et al. 2008;Wang and Gan 2009;Baby and Ramaprabhu 2010;Li et al.
2010;Shan et al. 2010). Iron magnetic nanoparticles have gained considerable
interest during the last few decades. ...

Immobilization of lactoperoxidase on Fe3O4 magnetic nanoparticles with improved
stability
Article
Full-text available
 * Dec 2019
 * BIOTECHNOL LETT

 * Seyed Ziyae Aldin Samsam Shariat
 * Mehrnaz Movahedi
 * Habibollah Nazem

Objective The study aimed to develop a facile and effectual method to increase
the stability of lactoperoxidase (LPO) by using its immobilization on Fe3O4
magnetic nanoparticles (Fe3O4 MNPs). Results The successful immobilization of
LPO on Fe3O4 MNPs was confirmed by using Fourier transform infrared spectroscopy
(FT-IR) and field emission scanning electron microscopy (FE-SEM). The Km values
of free LPO and LPO immobilized on Fe3O4 were 53.19, 72.46 mM and their Vmax
values were 0.629, 0.576 µmol/mL min respectively. The overall results indicated
that the stability of the immobilized LPO was significantly improved compared to
free LPO. The LPO immobilized on Fe3O4 (LPO– Fe3O4) retained 28% of the initial
activity within 30 days at 25 °C whereas the free enzyme lost its activity after
7 days at the same temperature. Moreover, evaluation of the thermal stability of
LPO at 75 °C determined the conservation of 19% of the initial activity of LPO
in the LPO– Fe3O4 sample after 60 min whereas the free enzyme lost its activity
after 5 min. Conclusions According to the present results, Fe3O4 magnetic
nanoparticles are suitable for the immobilization of LPO.
View
Show abstract
An Introduction to Magnetic Nanoparticles: From Bulk to Nanoscale Magnetism and
Their Applicative Potential in Human Health and Medicine
Chapter
 * Aug 2021

 * Assoc. Professor Costica Caizer
 * Dr. Shital Bonde
 * Mahendra Rai

The magnetism of magnetic nanoparticles is different from that of the
corresponding bulk magnetic material having the same chemical composition. The
different magnetic properties of magnetic nanoparticles are mainly due to their
small size, in the field of nanometers – tens of nanometers, which lead to a
different magnetic structure from the bulk, and the presence of surface effects
regarding the noncollinear arrangement of spins that is pronounced. At the same
time, with the sizes of magnetic nanoparticles of the order of nanometers, an
additional effect appears: superparamagnetism. As a result the magnetization of
small nanoparticles is no longer stable, but fluctuating at 180° under the
action of thermal activation. This behavior leads to the lack of hysteresis loop
in the magnetization of nanoparticles in the external magnetic field, and their
magnetization is similar to paramagnetics, according to the Langevin function.
All these aspects regarding the magnetism of nanoparticles, with multiple
applications in nano- and bionanotechnology, are presented and discussed in
these chapter compared with the magnetism of bulk magnetic material. Also, in
the chapter are presented the most important current biomedical applications of
magnetic nanoparticles: diagnosis and detection of diseases, smart drug delivery
system, therapeutic applications, and theranostic applications of
multifunctional magnetic nanoparticles.
View
Show abstract
Functionalized nanomaterials for environmental applications
Chapter
 * Jan 2021

 * Asit Baran Samui

Functionalized nanomaterials have been found to possess excellent properties.
Therefore, they can be gainfully utilized for environmental sensing and removal
of undesired materials. This chapter discusses the development of functionalized
magnetic nanoparticles (MNPs) for environmental sensing and removal of toxic
chemicals, including heavy metals and biological contaminants, from various
complex samples. The ongoing developments in the design and synthesis of
engineered nanomaterials afford high surface area-based function. Imparting a
magnetic nature to nanomaterials adds operational advantages, and adding
functionalization provides the ability to interact with surrounding chemicals
and species. This chapter focuses on the synthesis, functionalization, and
application of engineered MNPs for sensing and removing undesired materials and
species.
View
Show abstract
Selective, Agglomerate-Free Separation of Bacteria Using Biofunctionalized,
Magnetic Janus Nanoparticles
Article
 * Jul 2019

 * Reshma Kadam
 * Michael Maas
 * Kurosch Rezwan

This study presents a scalable method for designing magnetic Janus nanoparticles
which are capable of performing bacterial capture while preventing agglomeration
between bacterial cells. To this end, we prepared silica-coated magnetite Janus
nanoparticles functionalized with a bacteria-specific antibody on one side and
polyethylene glycol chains on the other, using the established wax-in-water
emulsion strategy. These magnetic Janus nanoparticles specifically interact with
one type of bacteria from a mixture of bacteria via specific antigen-antibody
interactions. Contrarily to bacterial capture with isotropically functionalized
particles, the bacterial suspensions remain free from cell-nanoparticle-cell
agglomerates owing to the passivation coating with polyethylene glycol chains
attached to the half of the magnetic nanoparticles pointing away from the
bacterial surface after capture. Selective magnetic capture of Escherichia coli
cells was achieved from a mixture with Staphylococcus simulans without
compromising bacterial viability and with an efficiency over 80 %. This approach
is a promising method for rapid and agglomeration-free separation of live
bacteria for identification, enrichment and cell counting of bacteria from
biological samples.
View
Show abstract
Extraction of Plasmid DNA by use of a magnetic maghemite-polyaniline
nanocomposite
Article
 * Mar 2019

 * Romário Silva
 * Bruna Maciel
 * Juan Carlos Medina Llamas
 * Celso de Melo

We describe the use of a hybrid magnetic nanocomposite (HMNC) for the extraction
and purification of plasmid DNA (pDNA) from Escherichia coli aqueous solutions.
The HMNC, which was synthesized via emulsion polymerization, was characterized
by transmission electron microscopy, scanning electron microscopy, UV-Vis
spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering
and magnetic measurements. The results confirmed the incorporation of
polyaniline (Pani) in its conducting form onto a core formed by the magnetic
iron oxide, with the hybrid particles presenting an average size of (95 ± 30) nm
and a saturation magnetization of 30 emu/g. The yield, purity and quality of the
pDNA purified by using the Pani HMNC were evaluated by UV-Vis spectroscopy,
agarose gel electrophoresis, and Polymerase Chain Reaction (PCR), respectively.
An average yield of ~ 6.9 g was obtained in the DNA extraction process, with
the collected material presenting a good purity (a ₳260/280 ratio in the 1.68 -
1.82 range) and an excellent quality, as confirmed by subsequent PCR assays.
Hence, this HMNC appears as a promising material for use in pDNA purification
protocols, and we suggest that this novel HMNC-based methodology can be of
general interest and find widespread application in different biomedical
procedures.
View
Show abstract
Magnetic plasmonic particles for SERS-based bacteria sensing: A review
Article
Full-text available
 * Jan 2019

 * Chaoguang Wang
 * Marco Massimiliano Meloni
 * Xuezhong Wu
 * Peitao Dong

This review describes recent advances in the use of magnetic-plasmonic particles
(MPPs) for bacteria detection by Surface-Enhanced Raman Scattering (SERS).
Pathogenic bacteria pollution has always been a major threat to human health and
safety. SERS spectroscopy has emerged as a powerful and promising technique for
sensitive and selective detection of pathogen bacteria. MPPs are considered as a
versatile SERS platform for their excellent plasmonic properties and good
magnetic responsiveness. Improved preparation method and typical
characterization technique of MPPs are introduced, focusing on the thin and
continuous metallic shell covering process. Consequently, the SERS-based sensing
methods for bacteria identification were discussed, including the label-free and
label-based methods. Finally, an overview of the current state of the field and
our perspective on future development directions are given.
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Show more

Preparation and characterization of carboxyl-group functionalized
superparamagnetic nanoparticles and the potential for bio-applications
Article
Full-text available
 * Jan 2007
 * J BRAZIL CHEM SOC

 * Zhi Shan
 * Wan-Shen Yang
 * Xu Zhang
 * Hui Ye

Neste trabalho, foi desenvolvido um método para preparação de nanopartículas
magnéticas monodispersas funcionalizadas com grupos carboxila. Maguemita de
dimensões nanométricas (y-Fe 2 O 3 , 7,0 ± 1,0 nm) foi sintetizada usando-se o
método de coprecipitação térmica e subseqüentemente coberta com grupos
funcionais por copolimerização em suspensão conduzida em uma etapa. Estudos de
espectroscopia de infravermelho com transformada de Fourier e análise
termogravimétrica confirmaram o sucesso da funcionalização dos grupos carboxila
na superfície dos nanocristais magnéticos. Esta superfície química torna
possível a purificação de DNA baseada em SPRI (imobilização reversível em fase
sólida). Assim, as nanopartículas foram empregadas para isolamento de DNA de
cultura de células bacterianas e os resultados demonstraram sua aplicabilidade
na preparação de DNA. In this work, a method was developed to prepare
monodispersed carboxyl-group functionalized magnetic nanoparticles. Nanosized
maghemite (y-Fe 2 O 3 , 7.0 ± 1.0 nm) was synthesized using thermal
co-precipitation method and subsequently coated with functional groups by
one-step suspension copolymerization. The Fourier transform infrared
spectroscopy study and thermogravitmetric analyses confirmed the successful
functionalization of carboxyl groups on the surface of magnetic nanocrystals.
The surface chemistry makes it possible for SPRI (solid phase reversible
immobilization)-based DNA purification. Thus the nanoparticles were employed to
isolate plasmid DNA from bacterial cell culture and the results demonstrated its
applicability in DNA preparation.
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Application of silica–magnetite nanocomposites to the isolation of ultrapure
plasmid DNA from bacterial cells
Article
 * Oct 2006
 * J MAGN MAGN MATER

 * Chen-Li Chiang
 * Ching-Shan Sung
 * Chuh-Yean Chen

The aim of this study was to develop a simple and rapid method for purification
of ultrapure plasmid DNA with high yields from bacterial cultures. Nanosized
superparamagnetic nanoparticles (Fe3O4) were prepared by chemical precipitation
method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen
atmosphere. Silica–magnetite nanocomposites were prepared by the method of acid
hydrolysis of tetraethoxysilane (TEOS) to coat the silica onto magnetite
nanoparticles. DNA was adsorbed to the support under high salt conditions, and
recovered directly in water for immediate downstream application, without the
need for precipitation. We demonstrated that a useful plasmid, pRSETB-EGFP,
encoding for the green fluorescent protein with T7 promoter, could be amplified
in Escherichia coli of DE3 strain. Up to approximately 43μg of high-purity
(A260/A280 ratio=1.75) plasmid DNA was isolated from 3ml of an overnight
bacterial culture. The eluted plasmid DNA was used directly for restriction
enzyme digestion, bacterial cell transformation and polymerase chain reaction
(PCR) amplification with success. The protocol, starting from the preparation of
bacterial lysate and ending with purified plasmid takes less than 8min. The
silica–magnetite nanocomposites deliver significant time-savings, overall higher
yields, lower RNA contamination, and better PCR amplification compared to
commercial available silica-based and other methods.
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Show abstract
Carboxyl Group (−CO2H) Functionalized Ferrimagnetic Iron Oxide Nanoparticles for
Potential Bio-Applications
Article
 * Sep 2004
 * J MATER CHEM

 * Shi Yu
 * Gan Moog Chow

A new approach to prepare surface-functionalized magnetic nanoparticles by
synthesis of poly(methacrylic acid) (PMAA) coated maghemite nanoparticles in
aqueous solution is reported. Maghemite (γ-Fe2O3) nanoparticles with an average
diameter of 8 ± 2 nm were fabricated and subsequently coated with PMAA by
emulsion polymerization. The FTIR study and thermal analysis confirmed the
chemical adsorption of methacrylic acid on the maghemite nanoparticle surface,
and suggested a symmetrical carboxylate bonding. The free carboxyl group of
PMAA, which was verified by FTIR spectroscopy and zeta potential measurement,
provided the site for immobilization of foreign molecules. The PMAA coated
maghemite nanoparticles were demonstrated as potential magnetically targeted
drug carriers by adsorbing an anti-cancer drug (carboplatin) via the ion–dipole
interaction between CO2− of PMAA and carboplatin.
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Show abstract
Screening of Chitosans and Conditions for Bacterial Flocculation
Article
 * Nov 2000

 * Sabina Strand
 * Marianne S. Vandvik
 * Kjell M Varum
 * Kjetill Østgaard

Chitosans with different chemical compositions and molecular weights have been
evaluated as flocculants of Escherichia coli suspensions. The flocculation
performance of chitosans at different conditions (pH, ionic strength) was
followed by residual turbidity measurements. For precise comparison, the
chitosan concentrations corresponding to 75% flocculated bacteria (x75) were
calculated from a mathematical function fitted to the measured data. At all
conditions, an increase in the fraction of acetylated units (FA) resulted in
lower x75 and thereby better flocculation efficiency. Especially the most
acetylated chitosans (FA 0.49 and FA 0.62) were excellent flocculants. An
increase in FA from 0.002 to 0.6 caused a 10-fold reduction in necessary
concentrations, at both pH 5 and 6.8. pH was a rather insignificant factor in
the range 4−7.4, further pH increase led to either increase of necessary doses
at low FA or sudden ceasing of flocculation at high FA. The chitosans
flocculated in a broad range of molecular weights, although an increase in
molecular weight was a favorable factor. Increase in ionic strength caused a
severalfold reduction in x75 for all chitosans and considerable broadening of
flocculation intervals.
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Show abstract
Rapid enrichment of leucocytes and genomic DNA from blood based on bifunctional
core–shell magnetic nanoparticles
Article
 * Apr 2007
 * J MAGN MAGN MATER

 * Xin Xie
 * Xiaorong Nie
 * Bingbin Yu
 * Xu Zhang

A series of protocols are proposed to extract genomic DNA from whole blood at
different scales using carboxyl-functionalized magnetic nanoparticles as
solid-phase absorbents. The enrichment of leucocytes and the adsorption of
genomic DNA can be achieved with the same carboxyl-functionalized magnetic
nanoparticles. The DNA bound to the bead surfaces can be used directly as PCR
templates. By coupling cell separation and DNA purification, the whole operation
can be accomplished in a few minutes. Our simplified protocols proved to be
rapid, low cost, and biologically and chemically non-hazardous, and are
therefore promising for microfabrication of a DNA-preparation chip and routine
laboratory use.
View
Show abstract
Molecular Cloning: A Laboratory Manual (3-Volume Set)
Book
 * Jan 2001

 * Sambrook J.F.
 * D. W. (Eds.) Russell

View
Plasmid DNA isolation using amino-silica coated magnetic nanoparticles (ASMNPs)
Article
 * Nov 2007

 * Xiaoxiao He
 * Hailing Huo
 * Kemin Wang
 * Jia Ge

A simple and efficient approach for the rapid isolation of plasmid DNA from
crude cell lysates has been described. The approach took advantage of the
amino-modified silica coated magnetic nanoparticles (ASMNPs) with positive zeta
potential at neutral pH and superparamagnetism under the external magnetic
fields. As a demonstration, the pEGFP-N3 plasmid has been concentrated and
isolated from the E. coli DH5alpha transformed with pEGFP-N3 plasmid through
electrostatic binding between the positive charge of the amino group of ASMNPs
and the negative charge of the phosphate groups of the plasmid DNA. Then the
pEGFP-N3 plasmid has been released easily and quickly from the pEGFP-N3
plasmid-ASMNPs complexes with 3M NaCl. The entire procedure could be carried out
by the aid of external magnetic fields in 15min and eliminate the need of
phenol, cesium chloride gradients or other noxious reagents and complexes
operation. Moreover, the pEGFP-N3 plasmid obtained by this approach retains
biological activity that can be suitable for restriction enzyme digestion and
cells transfection with expression of green fluorescence protein.
View
Show abstract
Isolation of Plasmid DNA Using Magnetite as a Solid-Phase Adsorbent
Article
 * Sep 1998

 * Martin J. Davies
 * James I. Taylor
 * Niki Sachsinger
 * Ian J. Bruce

View
Surface characterization and on-line activity measurements of microorganisms by
capillary zone electrophoresis
Article
 * Feb 1999
 * J Chrom B Biomed Sci Appl

 * Masaki Torimura
 * Shuichiro Ito
 * Kenji Kano
 * Teruhisa Ueda

Capillary zone electrophoresis (CZE) was applied to the electrophoretic
characterization for microorganisms. The electrophoretic peaks detected using
light scattering phenomena were characteristic of the microorganisms used. The
electrophoretic mobility (mu) evaluated by CZE was in good agreement with that
obtained by classical electrophoresis of microorganisms. The migration time was
reproducible and depended on the ionic strength (I). Analysis of the mu vs. I
relationship provided information regarding the charge density and the hardness
of the microbial cell surface. The redox enzymatic activity of microorganisms
was also evaluated by CZE using a running buffer containing a corresponding
substrate and an appropriate exogenous electron acceptor. A decrease in the
concentration of the electron acceptor due to microbial activity can be
simultaneously monitored during the electrophoretic process without significant
modification of the CZE instrument. Effects of some chemical treatments of
microbial cells were also studied using this technique.
View
Show abstract
DNA extraction using bacterial magnetic particles modified with hyperbranched
polyamidoamine dendrimer
Article
 * Apr 2003
 * J BIOTECHNOL

 * Brandon Yoza
 * Atsushi Arakaki
 * Tadashi Matsunaga

A cascading hyperbranched polyamidoamine dendrimer was synthesized on the
surface of bacterial magnetite from Magnetospirillum magneticum AMB-1 to allow
enhanced extraction of DNA from fluid suspensions. Characterization of the
synthesis revealed linear doubling of the surface amine charge from generations
one through five starting with an amino silane initiator. Furthermore,
transmission electron microscopy revealed clear dispersion of the single domain
magnetite in aqueous solution. The dendrimer modified magnetic particles have
been used to carry out magnetic separation of DNA. Binding and release
efficiencies increased with the number of generations and those of bacterial
magnetite modified with six generation dendrimer were 7 and 11 times
respectively as many as those of bacterial magnetite modified with only amino
silane.
View
Show abstract
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[ASSAYS ON COMPARING THE LOCAL CONCENTRATION OF HU PROTEIN IN THE DIFFERENT
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July 2005 · Молекулярная биология
 * O V Preobrazhenskaia
 * Elizaveta S Starodubova
 * Vadim Karpov
 * Josette Rouviere-Yaniv

HU, a nonspecific histone-like DNA binding protein is a major component of the
bacterial nucleoid. HU is referred to as an accessory factor for complex
protein-DNA assembly and as a protein involved in DNA compaction. In this study
we investigated in vivo HU binding along the different regions of E. coli
genome. For this purpose we used ChIP--in vivo formaldehyde crosslinking and ...
[Show full abstract] immunoprecipitation of protein-DNA complexes with
antiHU-antibodies. This technique allows to compare the local concentration of
HU protein in the different regions of E. coli genomic DNA. In this study we
analysed the HU-DNA crosslinking both in exponentially growing and stationary
phase of bacteria in the following regions of E. coli genome: oriC region,
promoter and structural regions of hupA and hupB genes coding two different
subunits of HU, and structural parts of dps and glgS genes which are active only
in stationary phase. Our results indicate that in exponentially growing E. coli
cells the local concentration of HU protein is uniform for all analysed regions
of genome and does not depend on their transcriptional status. The twofold
increase of local concentration of HU protein was also shown for all analysed
genome regions in the stationary phase cells.
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Data
Full-text available


DATASET S15

July 2004
 * Earl F Glynn
 * Paul Megee
 * Hong-Guo Yu
 * [...]
 * Jennifer L Gerton

SIMRUP2_187 Cy3 = ChIP cdc16-ts Smc3-6Myc in A364a; Cy5 = genomic DNA. (4.6 MB
XLS).
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DETECTION BY POLYMERASE CHAIN REACTION OF ALL COMMON MYCOPLASMA IN A CELL
CULTURE FACILITY

February 1995 · Pathobiology
 * J M Pruckler
 * Edwin W. Ades

The identification of cell cultures contaminated with organisms from the class
Mollicutes has led us to examine the effectiveness of polymerase chain reaction
(PCR) for detecting these organisms in genomic DNA. We developed a previously
identified nested PCR primer set and compared its ability to detect Mycoplasma
with that of a commercially available PCR kit for detecting Mycoplasma. We found
... [Show full abstract] that although the commercial system detected and
identified a few of the most common Mycoplasma species, the primer set (GPO-1,
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of the rare mycoplasma species previously encountered in tissue culture.
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[EVALUATION OF DETECTION OF M. TUBERCULOSIS IN CLINICAL SPECIMENS OF
TUBERCULOSIS BY DNA AMPLIFICATI...

March 1993 · Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi =
Chinese journal of tuberculosis and respiratory diseases
 * Y H Zhuang
 * G L Li
 * X G Zhang

The sensitivity of detection of M. tuberculosis genomic DNA were 1pg or 10-100
bacterial cell by PCR. Only M. tuberculosis, M. bovis and BCG were positive with
165 b.p band, but all other 14 mycobacterium and 10 bacteria of
non-mycobacterial tested, were negative. Of 75 sputum specimens of pulmonary
tuberculosis, the positive rate of PCR were 53.3%, culture method showed only
21.3%, fast-acid ... [Show full abstract] staining were 25.3%. 17
non-tuberculosis lung disease were negative in three methods. Of 58 tuberculosis
meningitis, the positive rate of PCR, the fast-acid staining and culture in
cerebrospinal fluid were 51.7%, 8.6%, 1.7% respectively. 30 non-tuberculosis
meningitis were negative in three methods. The results showed that DNA
amplification is a superior method with high degree of sensitivity and
specificity for rapid diagnosis of pulmonary tuberculosis and tuberculosis
meningitis.
Read more

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