Pathogenic Bacteria Detection by

Coupled Water Soluble Fluorescent

Nanocrystals
Doctoral Dissertation
Raquel Feliciano Crespo

Advisor: Dr. Oscar Perales Perez

 Background
 Challenges
 Motivation
www.mitre.org/publications/project
 Objectives
 Research Overview
 Methodology
 Case1: Cd-based QDs
 Case 2: Zn-based NPs
 Results
newscenter.lbl.gov
 Concluding Remarks
2
 Foodborne diseases have become a major public health
problem worldwide due to the significantly increased
incidence of foodborne diseases over the last 20 years
 The Center of Diseases Control in U.S. (including Hawaii and
Puerto Rico) report that 9.4 millions of Americans get ill for
foodborne pathogens, 128,000 are hospitalized and 3,000 die
annually.
 The most common foodborne pathogens:
 Escherichia coli: vomits, fever, bloody diarrhea
 Salmonella: Salmonellosis, typhoid fever, bloody diarrhea
 Listeria monocytogenes: Listeriosis, vomits, diarrhea
 Staphylococcus aureus: several bloodstream infection (skin,
internal organs)
 Commonly found: meat, poultry, dairy, vegetables, seafood
3
www.mxdepositphoto.com www.saludconsultas.org

Pathogenic
Remedial
Bacteria
Action

www.medgadget.com

Detection 4
o Pathogen detection method can be classified into the
following categories:
Method Advantage Limitation

Nucleic-acid based  High Sensitivity and • High equipment cost

(multiplex PCR) specificity • Assay time over 24 h
 High levels of • Limit of detection 105
reproducibility CFU/mL.

Immunological • Specific binding due • Time consuming

(ELISA) to antigen-antibody • Assays time over 1 – 5 days
• Large number of • Limit of detection for 103 to
samples 105 CFU/mL.

Biosensors • Low cost • Poor Photostability

(optical sensor) instrumentation • Limit of detection 104
• High sensitivity CFU/mL.

5
The foodborne pathogens usually exist within complex
biological matrixes, and the separation and enrichment
of the target pathogens are crucial steps for accurate
detection. Current pathogen detection method are time
consuming and lack detection sensitivity.

Therefore, a detailed and systematic study is necessary

for the detection of bacteria to reduce detection time,
improve sensitivity, and simplify the manipulation
process.

High fluorescent quantum dots (QDs) are proposed to

be used as fluorescence sensors for pathogen
detection.
6
  Unique optical properties
 Tuning
 Functionalization
 High photostability
 Water-stable

Why Cd and Zn-based QDs?

Cd-based:
 Highly Fluorescent
 Visible light excitation J Nanomed Res 2017, 5 (3)

Zn-based
 Highly Fluorescent
 Environmentally friendly 7
 Adapted from: R. Narang et al. Scientific Reports, 8, 5807 (2018)

OH

O
S
CdSe H2O
H2O
EDC

EDC
⎯NH
plasmids QDs
DNA
SSSS SSSS
H2O

Outer
EDC H2O
membrane H2O

EDC

9
R. Feliciano et al. Journal of Electronic Materials, Vol 47 (8), 2018
Main Objective
 Establish the applicability of Cd-based QDs and Zn-based NPs
as a rapid method for bacteria detection using Fluorescence
Spectrometry as the analysis instrumentation for the real-time
pathogenic bacterial detection.
Specific Objectives:
 Determine the optimal parameters conditions to the enhancement
of the detection limit of pathogen species in presence of selected
QDs.
 Establish a relation between bacteria concentration and
fluorescence intensity peak spectra of Cd-based and Zn-based
QDs with Salmonella, E. coli, Listeria monocytogenes and
Staphylococcus aureus.
 Assess the applicability of Cd-based QDs and Zn-based NPs as
optical sensors for detection of pathogens with detection limit
about 102 CFU/mL. 10
 Use of Nanocrystals for Enviromental
and Biomedical Applications

Aqueous Synthesis of Synthesis of ZnO

CdSe QDs (Cd/Se, NPs in Ethylene
Characterization
time, T) Glycol
(XRD, TEM/SEM,FT-IR,
NIR, UV-Vis, PL)

Functionalization Functionalization
with TGA with GSH

Bactericidal Coupling Calibration Sensing

Test QDs-Bacteria Curve Validation 11
Case Study 1:
Cd-based QDs

13
 Cadmium acetate, trisodium citrate,
(D.I.)

pH Adjustment (11.0)

t =30 min

0.0043 0.0085 0.0127 0.0169 0.0212

mM mM mM mM mM
CdSe
No-TGA Addition of thiolglycolic acid
(TGA)

t = 60 min
[TGA]

Addition of Selenide (0.1 mM) CdSe Forming Reaction:

t = 15 min

pH Adjustment (7.00)
Cd+2 + TGA (Cd-TGA)+2
(Cd-TGA)+2 + HSe - + OH- CdSe/TGA + H2O
Purification

CHARACTERIZATION

13
R. Feliciano; et al. Materials Research Society (MRS), 1449, (2012)
 (102) XRD Pattern HRTEM Image
(a) JCPDS: ( 99-101-0836) (b)
Intensity (a.u.)

(112)

(203)

10 nm
10 20 30 40 50 60 70 80

2 theta degree [TGA]: 0.0212 mM

Average crystallite size @ 7 nm
Lattice parameters “a”: a =4.13 Å ; c=6.95Å
CdSe bulk “a”: 4.29
CdSe bulk “c”: 7.01 Scherrer’s t  0.9
B cos 14
equation:
 I

I
e

I
d CdSe
Intensity (a.u.)

I
I
c

I
I

b I

I
CdSe

I
I

wavenumber (cm-1)

I
(a): 0.0043 mM, (b): 0.0085 mM, (c): 0.0127 mM, (d): 0.0169 mM, (e): 0.0212 mM

The presence carboxylic groups is suggested by the strong C=O

band around 1565 cm-1 whereas the O-H group appeared at 3000-
3300 cm-1.
15
 3.5
RCO2R
(combination
band) 3

CH2 2.5
(combination
band)
2
OH
(second

Log 1/R
1.5
overtone)
S-H
(first 1
overtone)

0.5

-0.5

-1

wavenumber (cm -1)

The combination band 4200 cm-1 corresponds to CH2 and the one
around 5100 cm-1 suggested the presence of RCO2R functional group.
The presence of a weak band at 5760 cm-1 can be assigned to the first 16
overtone of S-H group. (CdSe-TGA sample of 0.0212 mM)
 [TGA] max
mM (nm)
0.0043 536

Band Gap Energy 0.0085 457

Absorbance (a.u.)
0.00127 422
Estimation
0.00169 413

0.0043 mM: 2.28 eV 0.0212 398

0.0085 mM: 2.35 eV

0.0127 mM: 2.41 eV
0.0169 mM: 2.45 eV
0.0212 mM: 2.56 eV
(bulk CdSe: 1.74 eV)

wavelength (nm.)

[TGA]
UV-Vis of CdSe with different TGA-capped from 0.0046 to 0.0212 mM.
Estimate band gap energies were calculated from 2.3 to 2.6 eV,
respectively; As the concentration of TGA increases, the electron volts
17
of the estimate band gap decreases.
 CdSe, no TGA
Band gap energy:
FWHM: 48 nm
Bulk: 1.74 eV
Absorbance (a.u.)

CdSe Bare: 2.36 eV

CdSe/TGA: 2.56 eV
=490 nm

CdSe, TGA (0.0212 mM)

FWHM: 26 nm

Absorbance (a.u.)
300 350 400 450 500 550 600 650

wavelength (nm)
=400 nm

Excitation wavelength: 400 nm 300 350 400 450 500 550 600 650

wavelength (nm) 18
 Pathogens Detection in Presence of QDs
Gram -: Escherichia coli (Atcc: 35218), Salmonella typhimurium (Atcc: 14020)
Gram +: Staphylococcus aureus (Atcc: 29213), Listeria monocytogenes (Atcc: BAA-752)

Bactericidal Test Coupling of QDs UV VIS and Validation

in presence of and NPs with Fluorescence Method
QDs and NPs bacterial analysis Assays

Minimum Assures Optical Confirms

Inhibitory Coupling with properties Detection
concentration bacteria response Method 19
20
Resazurin Assays Qualitative Analysis of E. coli and S.
typhimurium
- + 500 750 1000 1250 1500 1750 2000 2250 2500

• Control -: nanomaterial, QDs E

• Control +: pathogen strain E
• The blue color represents no E
presence of bacteria growth. E
S
MIC MBC S
E. coli +2500 ppm ------ S
S. typhi 2500 ppm 2750 ppm S

MIC

21
https://www.creative-bioarray.com/support/resazurin-cell-viability-assay.htm
 Incubation at 37 C
overnight McFarland
Step 1 Standard 0.5
(1.5 x108 CFU/mL)

Step 2
Dilution
108 107 106 105 104 103 102

Step 3

Control 105 CFU + 100 ppm

Incubation at 37 C
Until mid-log phase

22
Fluorescence Intensity vs Incubation time (minutes) of
Escherichia coli (Bacteria concentration : 105 CFU/mL)
60 min

Fluorescence Intensity
Fluorescence Intensity (a.u.)

45 min
30 min

(a.u.)
0 20 40 60 80
Time (minutes)

400 450 500 550 600 650 700 750 800

wavelength (nm)

Excitation wavelength: 400 nm

[QDs]: 100 ppm
23
Fluorescence Intensity of E. coli Concentration coupled with
Cd-based QDs (Incubation time = 45 min); ([QDs]: 100 ppm)

Fluorescence Intensity
105 CFU
y = 104.4x + 486.6
104 CFU R² = 0.9676
Fluorescence Intensity (a.u.)

103 CFU

(a.u.)
102 CFU

0 2 4 6
Log Bacteria Concentration
(CFU/mL)

400 450 500 550 600 650 700 750

wavelength (nm)
24
 Fluorescence Intensity
105 CFU y = 27x + 476.5
R² = 0.9867
104 CFU

103 CFU
Fluorescence Intensity (a.u.)

(a.u.)
102 CFU

0 2 4 6
Log Bacteria Concentration
(CFU/mL)

400 450 500 550 600 650 700 750 800

wavelength (nm) 25
 I

I
1. EDC CdSe
H-EDC

I
I

OH

I
O
2. EDC H-EDC
S
CdSe H2O
ZnO H2O ZnO
EDC
I OH
I EDC
NH2
⎯NH
O

3.
S
I

CdSe

CdSe plasmids QDs H2O

DNA
SSSS SSSS
EDC
I

I
H2O ⎯NH
plasmids
SSSS SSSS
H2O H2O
I

EDC
4. Bacteria Concentration Correlation
HO
(PL) 2
EDC

H2O

H2 O
CdSe

CdSe
H2 O
⎯NH CdSe
H2 O CdSe
H2 O
ED
⎯NH ⎯NH
SSSS SSSS ⎯NH
H2 O SSSS H2 O

EDC
SSSS SSSS SSSS
CdSe
H2 O H2 O SSSS SSSS
CdSe H2 O
EDC H2 O
⎯NH H2 O
H2 O CdSe EDC ⎯NH H2 O EDC
CdSe CdSe EDC H2 O
CdSe
SSSS SSSS ⎯NH
H2 O
⎯NH SSSS SSSS ⎯NH
H2 O ⎯NH
CdSe H2 O
SSSS SSSS
EDC SSSS SSSS H2 O SSSS SSSS
⎯NH EDC SSSS SSSS
H2 O H2 O H2 O H2 O H2 O
EDC CdSe CdSe
EDC SSSS SSSS EDC
H2 O ⎯NH H2 O EDC
CdSe ⎯NH

EDC SSSS ⎯NH

SSSS SSSS SSSS
H2 O H2 O
SSSS SSSS
EDC H2 O EDC

EDC

10 2 10 3 10 4 10 5 45
• The fluorescence intensity with respect to the
bacteria concentration presents a linear
correlation which allow the determination of the
bacteria concentration for Cd-based QDs and Zn-
based NPs in aqueous environments.

• The detection limit of the coupling of Cd-based

and Zn-based QDs was 102 CFU/mL.

• The method could be validated using an unknown

sample of Salmonella typhimurium by the linear
equation obtained by the calibration curve of
Salmonella typhi coupling assays. 46
 Incubation 37 C
Step 4 1000
500 750
Control ppm ppm ppm Shaker (200 rmp)
Sample Preparation

After 2
hour
Step 5
Dilution

D D D D D D D D D D D D
1 2 3 1 2 3 1 2 3 1 2 3
Step 6 100 µL 100 µL 100 µL 100 µL
Plates Preparation

55
 Step 1 Remarks:
Select the sample concentration
to be evaluated. (Vf : 100 µL)

Step 2
Incubate for 24 hours at Columns A and B
37 ºC. control - : QDs or Nanoparticles
control + : Bacteria strain

Step 3 Add 30 µL of 0.02% Resazurin

Prepare resazurin solution, incubate for 2 hours at
solution 0.02% 37 ºC.

56
 First color change indicated the
Step 4 minimum inhibitory concentration
Results after two hours (MIC) and second column the
of incubation minimum bactericidal concentration
(qualitative measure) (MBC)

Step 5 Control
750 ppm 1000 ppm

Inoculate MIC and MBC Inoculate 20 µL of control, MIC and

(quantitative measure) MBC wells and incubate for 24 h at
37 ºC.
Control
750 ppm 1000 ppm

57