NANO BIOSENSORS FOR DETECTON OF PATHOGNS N FOOD

ROHIT M-16102038 JATIN KAPOOR-16102193

 INTRODUCTION
A FACILE AND SENSITIVE DETECTION OF
PATHOGENIC BACTERIA USING MAGNETIC
NANOPARTICLES

 The early detection of pathogenic microorganisms at the lowest

possible concentrations is, in particular, critical because
microorganism populations increase rapidly over time.

 So, significant advances in the rapid detection of food

poisoning bacteria have been achieved using functional
nanoparticles.

 The central idea of nanoparticle-based detection is the

selective separation of target bacteria by application of an
external magnetic field.
 Application of a magnetic field separates the particle–bacteria
complexes from solution, thereby enriching the concentration of the
bacteria and enabling the detection of target bacteria without a
culturing process.

 In this study, we developed a simple, rapid, and cost-effective

method for the detection of pathogenic bacteria in foods using
magnetic (superparamagnetic) nanoparticles (MNPs) and TiO2
nanocrystals (TNs).

 The MNPs enabled the rapid extraction of pathogenic target

bacteria under an external magnetic field, and TNs were used as
optical nanoprobes for spectroscopic detection.
NEGLIGIBLE CHANGES with pH were observed for the
absorption spectra of TN’s indicating that TN’s are mire suitable
than gold nanoparticles for the quantification of bacterial
concentration
PREPARTION OF Fe3O4 MAGNETIC
NANOPARTICLES
4 mmol FeCl3, 12 mmol urea, and 8 mmol sodium citrate were
dissolved in 80 mL DI water.

Then 0.6 g polyacrylamide (7.5 g L—1) were added with vigorous

stirring.
The solution was transferred to a 100 mL Teflon-lined autoclave,
which was sealed and maintained at 200 ○C for 10 h
.

After the solution had cooled to room temperature, the precipitate

was collected by applying an external magnetic field.
The separation and washing steps were repeated several times using
water and absolute ethanol.

The resulting magnetic nanoparticles were further functionalized with

glutaraldehyde to form, respectively, amine groups and amine-reactive
crosslinkers on the surfaces.

Monoclonal antibodies toward Salmonella were then immobilized onto

the MNPs.
PREPARATION OF TiO2 NANOCRYSTALS (TN’s)
0.2 mL tert-butylamine were dissolved in 20 mL DI water, and the
solution was transferred to a 60 mL Teflon-lined stainless-steel
autoclave.

TTIP (0.3 g) and 2 mL oleic acid were dissolved in 20 mL anhydrous

toluene, and the solution was transferred to the autoclave.

The autoclave was then sealed, heated to 180 ○C for 12 h, and then
cooled to room temperature.
The crude TN solution was precipitated with methanol and isolated
by repeated centrifugation.

The purified TNs were finally redispersed in chloroform. An oil-in-

water micro-emulsion was formed, and the evaporation of
chloroform under heating (50 ○C ,30 min) resulted in the transfer of
TNs to the aqueous phase.

The resulting TNs were treated with glutaraldehyde, followed by

immobilization of polyclonal antibodies toward Salmonella on the TN
surfaces.
 Detection of Salmonella bacteria\

A schematic illustration of the pathogenic bacteria detection method using magnetic

nanoparticles and optical nanoprobes.
 Salmonella bacteria cultured in nutrient broth
and collected by centrifugation at 6000 rpm

 Samples prepared at conc. of 108 to 102 cfu mL- 1

in PB buffer and milk.

 100 μL of the antibody- immobilized MNPs

added to 10 ML OF SAMPLES

 The solutions incubated for 20 min with gentle

agitation

 The MNP–Salmonella complexes were

magnetically separated using a permanent
magnet and washed with buffer.

 Complexes re- suspended in 100 mL of a TN

solution (20 μg mL-1)

 Incubation for 20 min with gentle agitation.

 Magnetic separation of the MNP– Salmonella–

TN complexes.

 Spectra obtained for the unbound TN solution

using a UV- Vis spectrometer.
 Results and Discussions
The binding of antibody-conjugated TNs and MNPs to Salmonella was
investigated using TEM(Transmission electron microscopy).

Figure. TEM images of a Salmonella bacterium after binding to (a) bare MNPs (b)
antibody-immobilized MNPs.
UV–vis absorption spectroscopy is the measurement of the attenuation of a beam of
light after it passes through or reflects from a sample surface.

Figure below shows the UV-Vis absorbance spectra for various

concentrations of Salmonella ranging from 102 to 108 cfu mL-1.

The absorption spectra of unbound TiO2 nanocrystals in a buffer solution

(a) or in milk (b).The inset shows that the relative intensity at 230 nm is
proportional to the logarithmic concentration of Salmonella.
Advantages of this detection method:

 Immuno-sensing based on the light absorption of TNs

is more economic and less hazardous than sensing
methods based on fluorescence nanoparticles.

 Because the absorption intensity increased with

decreasing Salmonella concentration, this assay is
suitable for detecting low concentrations of Salmonella.

 Detection scheme is relatively insensitive to other

compositions in the sample matrix.
 Conclusion

 The increasing incidence of food poisoning from

microorganisms is a vital public health concern. The
early detection of pathogenic microorganisms at the
lowest possible concentrations is critical.

 Nano-particle based detection is a very efficient way

to rapidly detect the presence of pathogens in food.
Many new nano-biosensors are being researched
upon in order to detect the contamination in food.