Supporting Information

A Self-Quenching-Resistant Carbon Nanodot Powder With Multicolor

Solid-State Fluorescence for Ultra-Fast Staining of Various Representative

Bacterial Species within One Minute

Yongqiang Zhang,a,b

Chunfeng Li,c,d,e

Yi Fan,*a Chengbin Wang,

d Ruifu Yang,

c Xingyuan Liu*

a

and Lei Zhou*c

a State Key Laboratory of Luminescence and Applications, Changchun Institute of Optics, Fine

Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China

b University of Chinese Academy of Sciences, Beijing 100049, China

c Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity,

Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China

d Department of Clinical Laboratory, Chinese People’s Liberation Army General Hospital,

Beijing 100853, China

e Department of Blood Transfusion, The Second Hospital of Shandong University, Jinan 250033,

China

* E-mail: fanyi@ciomp.ac.cn (Y. Fan), liuxy@ciomp.ac.cn (X. Liu), ammszhoulei@aliyun.com

(L. Zhou)

Contents of supporting information:

1. Fig. S1 The process procedures in microwave synthesis of (a) SFCDs and (b) SNCDs.

2. Table S1. Surface functional groups of SFCDs identified by FT-IR spectra.

3. Fig. S2 The full scan XPS spectra of SFCDs and SNCDs.

4. Table S2. Lifetimes of SFCDs and SNCDs.

5. Fig. S3 Absorption and PL spectra of SNCD powder.

Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2016

2

6. Table S3. Comparisons between SFCDs and SNCDs.

7. Fig. S4 Fluorescence intensities and PL decay curves of SFCD solutions with different

concentrations.

8. Fig. S5 Fluorescence intensities and PL decay curves of SNCD solutions with different

concentrations.

9. Fig. S6 Fluorescence stability of SFCDs on a stained smear and under lucifuge storage.

10. Fig. S7 Confocal fluorescence microscopy images of SFCD-stained bacteria.

11. Table S4. Comparisons of our work with other 12 previous studies.

12. Fig. S8 A comparison of the staining effect on Bacillus anthracis endospores using the 1

min-SFCD-SSM and 1 min-SFCD-LIM.

13. Fig. S9 A comparison of the staining effect on intact Escherichia coli cells and Escherichia

coli cell debris.

14. Fig. S10 One minute staining effect of Staphylococcus aureus bacteria samples on the

dependence of concentration of SFCD solution.

15. Fig. S11 Graph of one minute effect of Staphylococcus aureus bacteria samples on the

dependence of concentration of SFCD solution.

16. Fig. S12 The PL excitation (PLE) and PL spectra of SFCD solution (100 mg·mL−1

).

17. Fig. S13 The lay scanning images of a Hela cell.

18. Fig. S14 The lay scanning images of an Escherichia coli cell.

19. Fig. S15 The magnification image of an Escherichia coli cell.

3

Fig. S1 The process procedures in microwave synthesis of (a) SFCDs and (b) SNCDs.

Table S1. Surface functional groups of SFCDs and SNCDs identified by FTIR spectra.

Peak and band The corresponding bond vibration

3700-3100 cm-1

νN-H and νO-H

3100-2500 cm-1

νO-H

1720 cm-1

νC=O in C=O in carboxylate

1670 cm-1

νC=O in COOH

1575 cm-1

δN-H

1450 cm-1

δCH2

1350 cm-1

vas

C-O in C-O-C in carboxylate

1208 cm-1

vsC-O in C-O-C in carboxylate

4

Fig. S2 The full scan XPS spectra of SFCDs and SNCDs.

Fig. S3 Absorption (ABS) and PL spectra of SNCD powder in the excitation wavelength range of

300-540 nm.

5

Table S2. Lifetimes of SFCDs and SNCDs.

Sample τ1 (ns)/ proportion τ2 (ns) / proportion χ2 τavg (ns)

SFCDs 3.29 / 18.53% 10.43 / 81.47% 1.006 9.11 ns

SNCDs 0.74 / 74.68% 3.44 / 25.32% 0.990 1.42 ns

Fig. S4 (a) Fluorescence intensities and (b) PL decay curves of SFCD solutions with different

concentrations (“I” is the current fluorescence intensity, and “I0” is the fluorescence intensity

under the concentration of 0.1 mg/mL).

Fig. S5 (a) Fluorescence intensities and (b) PL decay curves of SNCD solutions with different

concentrations (“I” is the current fluorescence intensity, and “I0” is the fluorescence intensity

under the concentration of 0.1 mg/mL).

6

Table S3. Comparisons between SFCDs and SNCDs.

Sample SFCDs SNCDs

Synthetic method Microwave Microwave

PL spectra in solid state

Quantum yield in solid

state 40% 1%

PL spectra in aqueous

solution

Quantum yield in

aqueous solution 43% 11%

Average particle size 2.3 nm 5.8 nm

Size distribution range 1-5 nm 3-11 nm

Solubility/Dispersibility Instant dissolve in water without

stirring and ultrasonic

Need 10 min or more time to

completely dissolve in water with

stirring and ultrasonic

7

Fig. S6 Fluorescence stability of SFCDs on a stained smear and under lucifuge storage. (a)

Confocal fluorescence microscopy images of 1 min-SFCD-SSM-stained Staphylococcus aureus

taken on the day after staining. (b) Confocal fluorescence microscopy images of the same smear

taken on the eighth day after staining and following lucifuge storage. In the images: upper-left,

405 nm light excitation; upper-right, 488 nm light excitation; lower-left, bright field illumination;

lower-right, overlay of the previous three images.

8

Staining

classification

Morphological

classification Geneus Species

Confocal fluorescence microscopic

image of SFCD-stained bacteria

Gram positive

bacterium

Coccus

Staphylococcus Staphylococcus

aureus

Streptococcus Streptococcus

pneumoniae

Enterococcus Enterococcus

faecalis

Bacillus Bacillus

Bacillus

anthracis

vegetative cell

endospore

Bacillus subtilis

vegetative cell

endospore

9

Staining

classification

Morphological

classification Geneus Species

Confocal fluorescence microscopic

image of SFCD-stained bacteria

Clostridium Clostridium

sporogenes

vegetative cell and endospore

Listeria Listeria

monocytogenes

Gram negative

bacterium

Coccus Neisseria Neisseria

meningitidis

Bacillus

Yersinia Yersinia pestis

Pseudomonas Pseudomonas

aeruginosa

Klebsiella Klebsiella

pneumoniae

Escherichia Escherichia coli

10

Staining

classification

Morphological

classification Geneus Species

Confocal fluorescence microscopic

image of SFCD-stained bacteria

Spirillum Vibrio Vibrio cholera

O1

Acid-fast

bacterium Bacillus Mycobacterium

Mycobacterium

smegmatis

Fig. S7 Confocal fluorescence microscopy images of SFCD-stained bacteria. All bacteria can be

classified according to staining methods, including the Gram-positive bacteria (especially the

Bacillus anthracis endospore, with thick capsule), Gram-negative bacteria (especially Neisseria

meningitidis and Klebsiella pneumoniae, with lipids and polysaccharides contained in their cell

walls), and acid-fast bacteria (especially Mycobacterium smegmatis, with a large amount of lipid

contained in its cell walls). All samples were prepared using the 1 min-SFCD-SSM. In the

images of SFCD-stained bacteria: upper-left, 405 nm light excitation; upper-right, 488 nm light

excitation; lower-left, bright field illumination; lower-right, overlay of the previous three images.

11

Table S4. Comparisons of our work with other 12 previous studies.

Sample Synthesis

Fluorescenc

e (solid

state and

solution)/

FQY

Bacterial species Staining

classification

Staining

method

Staining

time

SFCDs

microwav

e synthesis

for 3 min

Solid state/

40 % and

solution/ 43

%

Bacillus anthracis

(vegetative cell and

endospore), Bacillus subtilis (vegetative cell and

endospore), Clostridium sporogenes (vegetative cell

and endospore), Listeria

monocytogenes,

Enterococcus faecalis,

Staphylococcus aureus,

Streptococcus pneumoniae,

Gram positive

bacterium

SSM

(applica

ble to

LIM)

1 min

Neisseria meningitidis,

Yersinia pestis,

Pseudomonas aeruginosa,

Klebsiella pneumoniae,

Escherichia coli, Vibrio cholera O1

Gram

negative

bacterium

Mycobacterium smegmatis Acid-fast

bacterium

CDs1

Hydrother

mal

heating

150 °C for

12 h

Solution/4.2

7 %

Pseudomonas aeruginosa Gram

negative

bacterium LIM 1-6 h

Mycobacterium

tuberculosis

Acid-fast

bacterium

Man-CQ

D2

Heating at

180 °C for

2 h

Solution/

8.8 % Escherichia coli

Gram

negative

bacterium

LIM 1 h

Man-CQ

D3

Heating at

180 °C for

2 h

Solution/

9.8 % Escherichia coli

Gram

negative

bacterium

LIM 1 h

Amphiphi

lic Carbon

Dots4

Hydrother

mal

heating for

1.5 h.

Solution/

None Pseudomonas aeruginosa

Gram

negative

bacterium

LIM 12 h

C-dots5

Hydrother

mal

heating at

170 °C for

12 h and

dialysis

for 12 h

Solution/

7.6 % Pseudomonas aeruginosa

Gram

negative

bacterium

LIM 1-6 h

Carboxyl-

modified

CDs6

Hydrother

mal

heating at

200 °C for

5 h

Solution/ 76

% Salmonella typhimurium

Gram

negative

bacterium

LIM 1 h

12

Amphiphi

lic carbon

dots7

Heating at

90 °C for

5 h

Solution/

4.7 %

Escherichia coli,

Salmonella typhimurium,

Pseudomonas aeruginosa

Gram

negative

bacterium LIM 3 h

Bacillus cereus Gram positive

bacterium

CDs8

Hydrother

mal

heating at

120 °C for

180 min

Solution/

6.9 % Escherichia coli

Gram

negative

bacterium

LIM 1-6 h

CQDs9

Acid

overnight

treatment

Solution/

None

sewage water bacteria (no

name) N/A LIM

30-40

min

C-dots10

Hydrother

mal

heating at

125, 150

and 170°C

for 12 h

and

dialysis

for 12 h

Solution/

7.0 % Bacillus subtilis

Gram positive

bacterium LIM 1-6h

CQD11

Reflux at

90 °C in

the

presence

of 5 mL of

conc.

sulfuric

acid for 4

h

Solution/

11.8 % Escherichia coli

Gram

negative

bacterium

LIM 24 h

DNA-CD

s12

Hydrother

mal

heating at

180 °C for

12 h

Solution/

None Escherichia coli

Gram

negative

bacterium

LIM 6 h

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Fig. S8 A comparison of the staining effect on Bacillus anthracis endospores using the 1

min-SFCD-SSM and 1 min-SFCD-LIM. (a) Confocal fluorescence microscopy images of

Bacillus anthracis endospore with 1 min-SFCD-SSM (in which Bacillus anthracis endospores

were fixed on a slide and then stained with a SFCD solution for 1 min. (b) Confocal fluorescence

microscopy images of Bacillus anthracis endospore with 1 min-SFCD-LIM (in which a Bacillus

anthracis endospore suspension was incubated with a SFCDs solution for 1 min, and then

stained endospores were separated with SFCDs by centrifugation and fixed onto a slide). In the

microscopy images: upper-left, 405 nm light excitation; upper-right, 488 nm light excitation;

lower-left, bright field illumination; lower-right, overlay of the previous three images.

14

Fig. S9 A comparison of the staining effect on intact Escherichia coli cells and Escherichia coli

cell debris. (a) Confocal fluorescence microscopy images of intact Escherichia coli cells stained

using the 1 min-SFCD-SSM. (b) Confocal fluorescence microscopy images of Escherichia coli

cell debris stained with 1 min-SFCD-SSM. In the images: upper-left, 405 nm light excitation;

upper-right, 488 nm light excitation; lower-left, bright field illumination, lower-right, overlay of

the previous three images.

15

10 μm

50 9070 8060

SFCDs Concentration(mg/ml)

λex

: 4

05n

mλex

: 4

88n

m

10 μm

100 110 120 130 140

λex

: 4

05n

mλex

: 4

88n

m

Fig. S10 1 min staining effect of Staphylococcus aureus bacteria samples on the dependence of

concentration of SFCD solution. The concentration has a great influence on staining effect. A

suitable concentration provides an ideal osmotic pressure, and is one of the necessary

requirements for the rapid infiltration process of SFCDs into bacterial cell (1 min-SSM). In this

study, the optimized concentration of SFCDs saline solution is 100 mg·mL−1

.

16

Fig. S11 Graph of 1 min staining effect of Staphylococcus aureus bacteria samples on the

dependence of concentration of SFCD solution.

Fig. S12 The PL excitation (PLE) and PL spectra of SFCD solution (100 mg·mL-1

) at excitation

wavelengths in the range of 320-560 nm.

17

Fig. S13 Layer scanning photographs under LSCM of a Hela cell based on 1 min-SFCD-SSM

staining, (a) excited by 405nm, (b) excited by 488nm, (c) overlay of the images excited by 405

and 488nm, simultaneously, (d) bright field illumination (control group), and (e) overlay of the

previous the simultaneous excitation under 405nm, 488nm and bright field. These scanning were

operated from top to down and the interval was 2 μm. Scale bar: 10 μm.

Fig. S14 The lay scanning images of Escherichia coli (from 1 to 12), the scale bar is 10 μm.

18

Fig. S15 The magnification image of an Escherichia coli cell, the scale bar is 10 μm.