NANO-BIOCOMPOSITE FOR FAST BACTERIAL DETECTION

Rodrguez-Martnez C,1 Lobaina-Rodrguez T,1 Zhurbenko R,1 Alfonso-Valds I,1 Martins-Gomes AD,2 de Lacerda-Gontijo SM,3
Suarez-Pearanda D,2 Sinisterra RD,2 Segura-Corts ME4
1 Research Direction, Centro Nacional de Biopreparados, Cuba. 2 Chemestry Department, Universidade Federal de Minas
claudio@biocen.cu Gerais, Brazil. 3 Physiology Department, ICB, Universidade Federal de Minas Gerai,s Brazil. 4 Restorative Dentristy Faculty,
Universidade Federal de Minas Gerais, Brazil
INTRODUCTION: New methods in diagnostic microbiology have being designed based on nanotechnology. Few of them have reached the market due to they high cost. BioCen and CNIC developed a new method for the
obtainment of nano-biocomposites for bacterial and yeast detection in clinical and environmental samples. The objective of this research work was the evaluation of a new nano-biocomposite for the fast detection of E.
coli as a model of clinical infection, based on the combination of an hydroxyapatite nanostructure with fluorogenic enzymatic markers and a mixture of growth promotion substances of biological origin.
MATERIAL AND METHODS: Different protein hydrolysates, growth factors and molecular enzymatic activity markers were combined with calcium phosphate nano-particles to develop a new method for fast bacterial
detection. The obtained composite was characterized by TEM, EDS, FTIR, TGA and other different characteristic were measured, such as Z potential, particle size, loss on drying, water loading capacity, pH and
antibacterial activity. The influence of sample concentration and volume, hydration of the composite and the detection method were established. The bacterial presence was tested by visual or spectrofluorometric
detection under 366 nm UV light. The composite (HAP-S/CCL) was obtained by combining calcium phosphate nano particles with a nutrient mixture which promotes the fast bacterial multiplication containing 4 different
protein hydrolysates,
4-methylumbellipheryl--D-glucuronide as a biomarker and other bacterial growth factors. The possible inhibitory properties of the nano-biocomposite was measured by observing growth
inhibition zones in Mueller Hinton Agar. The time necessary for fluorescence detection was observed by naked eye under UV light (365 nm) and by spectrofluorometric method variying the concentration and inoculum of
bacterial suspension and the prehydration of the nano-biocomposite before the growth observation.
RESULTS AND DISCUSSIONS: The hydroxyapatite nanostructure coupled with the enzymatic biomarker and the growth promotion mixture of biological substances (HAP-S/CCL) contains a calcium to phosphor rate
(Ca:P) of 1,33 and also contains Mg- 0,09 %, Al-<0,20 %, Fe- 0,02 % and Pb- <0,20 %. TEM analysis showed that nanoparticles are well integrated in a highly rough surface structure and some nano-particles of the
nutritive composition are attached and distributed on the surface of the composite. XRD spectrum showed an appropriate crystalline structure and TGA demonstrated a high temperature stability of the tested nanobiocomposite. The obtained nanostructure showed173,2 nm average particle size; 20,67 mV zeta potential; 1,43 % loss on drying; an average 28 % loading capacity, 7,2 pH and did not showed any antibacterial activity
against Gram-negative and
Gram-positive bacteria. The developed procedure allowed E. coli detection as a model, by contacting the composite with bacterial suspensions, incubating for a reduced time (60 to 90 min)
and detecting the fluorescence under UV light or by measuring the signal in an automatic reader. An intense fluorescence was observed after incubating for a maximum of 90 min. It was demonstrated that HAP-S/CCL
did not show any inhibitory effect on bacteria. It was possible to detect the presence of E. coli in a 0,4 mL sample at a 3 x 108 CFU/mL concentration in a period of 1:30 h with only 0,1 g of the nano-biocomposite. The
previous hydration of the nano composite before the inoculation and the detection of the fluorescence in a spectrofluorometer allowed to detect E. coli in only 1 h.

Parameter

Time (h)

Mean and Std. Dev.

Loss on drying (%)

1.430.60

27.941.84

30.214.41

29.511.40

Load capacity (%)

Table 1. Loss on drying of the HAP-S and its loading capacity

when imbibing from 1 to 3 h in water at 25 C.
Figure 1. Imgens of the Scan Surface Microscopy (A) Agregates of the HAP-S
nanopartcles, (B) Bio-nanocomposite HAP-S/CCL.

Figure 3. EDS of the agglomerate of calcium phosphate

nano-sized particles. (a): HAP-S sample. (b): HAP-S
elemental composition plot. (c): HAP-S elemental
composition. (d): HAP-S/CCL sample. (e): HAP-S/CCL
elemental composition plot. (f): HAP-S/CCL elemental
composition.

Fig. 4. FTIR spectra. (a): HAP-S.

(b): HAP-S/CCL.

Fig. 6. X-ray difraction spectra. (a): HAP-S. (b): HAP-S/CCL.

Figure 9. Hydration of the HAP-S/CCL. Influence of the water volume

added to the bio-nanocomposite (0.1 and 0.2 ml), hydration time (1 and
2 h) and inoculum volume (0.1 and 0.2 ml) on the fluorescence
detection time.

Figure 2. Particle size distribution by intensity and - potential. (a)

Particle size plot of the nutritive composition with the fluorogenic
substrate (CCL). (b) Particle size plot of HAP-S nano-sized
elemental particles. (c) Particle size plot of HAP-S/CCL composite.
(d) Mean particle size, percentage distribution and -potential of
tested materials.

Fig. 5. Thermal stability curve of HAP-S

and the composite HAP-S/CCL, with
emphasis in the temperature range of up
to 200 C.

Figure 7. Inhibitory effect of HAP-S nanoparticles at different

concentrations (clockwise) : 0.05 (1); 0.5 (2); 1 (3); 5 (4); 10 (5); 15 (6) and
20 (7) % m/v against: E. coli (ATCC 25922), P. aeruginosa (ATCC 27853),
E. faecalis (ATCC 29212) and S. aureus (ATCC 25923). Centered circles at
the top
(c) show inhibition halos against gentamicine, amikacine,
sulphamethoxazole-trimethoprim and chloramphenicol respectively as
ppositive controls.

Figure 10. Influence of the inoculum volume of E. coli (0.1; 0.2

and 0.3 ml) and the concentration (3 x 106 y 3 x 108 CFU/ml) on
the fluoresecence detection time.

Figure 8. Influence of different nanocomposites

(HAP-S/CCL
y HAP-S/MUG) and inculum volumes (0.2 and 0.4 ml) on the
fluorescence detection time and after 24 incubation. NF: no
fluorescence was observed after 24 h incubation time.

Figure 11. Spectrofluorometric and visual detection of the fluorescent signal

from 1 to 6 h incubation. (a): Imagen of spectrofluorometric detection. (b):
Imagen of the visual detection in tubes.

CONCLUSIONS: The bio-nanocomposite HAP-S/CCL is able to detect E. coli in a reduced 1 h time period and shows advantages such as simplicity in handling easy detection procedure and
low cost.