Fluorimetric detection of Candida albicans using cornstalk N-carbon

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Fluorimetric detection of Candida albicans using cornstalk N-carbon quantum dots modified with amphotericin B Dayu Yu, Ling Wang, Huanyu Zhou, Xiaojun Zhang, Lei Wang, and Nan Qiao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00131 • Publication Date (Web): 22 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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Bioconjugate Chemistry

1

Fluorimetric detection of Candida albicans using cornstalk N-carbon

2

quantum dots modified with amphotericin B

3

Dayu Yu 1, 2, *, Ling Wang 1, 2, Huanyu Zhou 1, 2, Xiaojun Zhang 1, 2, Lei Wang 1, 2, Nan

4

Qiao 1, 3, *

5

1

6

Province, Northeast Electric Power University, Jilin 132012, China

7

2

8

China

9

3

Sci-Tech Center for Clean Conversion and High-valued Utilization of Biomass, Jilin

School of Chemical Engineering, Northeast Electric Power University, Jilin 132012,

School of Civil Engineering and Architecture, Northeast Electric Power University,

10

Jilin 132012, China

11

*

(Yu D.Y.) E-mail: [email protected]; (Qiao N.) Email: [email protected]

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

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Due to the advanced fluorescence property of N-carbon quantum dots (N-CQDs), a

14

new method to detect pathogenic fungi by newly synthesized cornstalk N-CQDs

15

modified with water-soluble amphotericin B (N-CQDs@AmpB) was developed.

16

Specifically, N-CQDs with blue fluorescence were initially synthesized according to a

17

previous report and modified with amphotericin B on their surfaces. Subsequently, the

18

as-prepared N-CQDs@AmpB was used to detect Candida albicans, exhibiting a linear

19

range of 2.60 x 105 to 1.99 x 108 cfu/mL and a detection limit of 1124 cfu/mL.

20

Compared with other common methods, the method largely shortened the detection

21

time and enabled the process to be performed with minimal interference from complex

22

samples such as beef sausage. The high cost of water-soluble amphotericin B may

23

hamper

24

N-CQDs@AmpB. Thus, alcohol-soluble amphotericin B was used in subsequent

25

experiments, confirming its potential to broaden avenues for the detection of fungi.

26

Keywords: N-CQDs, Amphotericin B, Candida albicans, Fluorimetric detection,

27

Pathogenic fungus, Cornstalk

the

large-scale

application

of

the

new


detection

method

using

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INTRODUCTION

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The fungus Candida albicans is well known as a virulent pathogen and can cause

30

diverse diseases such as cutaneous candidiasis, coleitis, angular cheilitis, pneumonia

31

and even enterogastritis

32

treatment, so C. albicans infection has been considered to be one of the most serious

33

risks to public health worldwide. C. albicans grows well at physiological temperature

34

(37°C) and pH (7.4) 4 and exists in the oral cavity, intestinal tract and respiratory tract.

35

Hence, for human safety, the quantitative detection of C. albicans is of critical

36

importance. In recent years, various efforts have been devoted to exploring convenient

37

and accurate approaches for identifying fungi

38

drawbacks. On the basis of routine fungal culture and isolation technology, fungal

39

enrichment in medium followed by plate counting has served as the traditional method

40

8,

41

Another common detection technique is real-time fluorescent quantitative PCR 9.

42

However, the disadvantages of requiring highly skilled operators and expensive

43

equipments have limited wide applications of this approach. Consequently, traditional

44

assays towards pathogens are obviously defective, leading to the necessity of

45

developing rapid and sensitive strategies.

1-3.

Such infections can be lethal with improper medical

5-7,

but each of them has shown its own

but it is obviously limited by being time consuming and having low sensitivity.

46

Carbon quantum dots (CQDs) are a new type of nanometer-sized spherical carbon

47

fluorescent material and have received extensive attention in recent years due to their

48

unique properties, including high photostability

10, 11

and good biocompatibility


12, 13.


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14

49

Thus, these materials have been applied in a variety of fields, such as for biosensing

50

and fluorescent probes

51

ablation

52

abundant chemicals, such as strong acids and base, are necessary in some synthesis

53

methods. Hence, to reduce the use of hazardous chemicals, green approaches are

54

desirable, such as hydrothermal carbonization

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materials. Natural products containing sugarcane bagasse

56

be suitable starting materials for the fabrication of CQDs. Cornstalk is one of the main

57

natural products in agriculture and could be a green carbon source of CQDs due to its

58

high carbohydrate content 21. In addition, the fluorescence intensity and stability of the

59

synthesized CQDs could be improved in applications by doping with ethanediamine to

60

acquire N-CQDs, and the synthesis is rapid 22.

16,

15.

CQDs can be generated by many methods, including laser

electrochemical methods

17,

and wet chemical methods

19,

18.

However,

using natural sources as the starting 20

have been demonstrated to

61

In recent years, a combination of N-CQDs and aptamers or antibiotics has been

62

proposed as a novel fluorescence probe for the sensitive quantitative detection of

63

bacteria. Wang

64

conjugated aptamer allowed it to detect bacteria with a detection limit of 50 cfu/mL.

65

However, compared with bacteria, no suitable aptamers were found to be combined

66

with N-CQDs for detecting fungus. Zhong

67

detect gram-positive bacteria such as Staphylococcus aureus, and the detection limit

68

was 9.40 x 104 cfu/mL with obvious specificity. Amphotericin B is a polyene antifungal

69

antibiotic, a class of antibiotics with strong activity against most fungi such as C.

23

suggested that the excellent fluorescence intensity of the N-CQD

24

used CQDs modified with vancomycin to


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70

albicans 25, Cryptococcus neoformans 26, and Blastomyces dermatitidis 27. Formulations

71

of this compound have been widely used in antimicrobial therapy 28 and can be divided

72

into two categories: amphotericin B deoxycholic acid sodium (water-soluble) and

73

amphotericin B (alcohol-soluble). Amphotericin B can also be combined with C.

74

albicans because of the receptors on the cell membrane of this fungus

75

Simultaneously, amphotericin B could treat infections caused by fungus. Because

76

amphotericin B contains many hydroxyl and carboxyl groups, it has been easy to

77

combine this drug with CQDs or N-CQDs. Thus, it was possible to detect C. albicans

78

by N-CQDs modified with amphotericin B.

25.

79

It has been reported that amphotericin B linked with one-dimensional carbon

80

nanotubes could be used for drug delivery and therapy of diseases. For example,

81

amphotericin B linked to carbon nanotubes could reduce the cytotoxicity of the drug

82

and improve its transmission route

83

reported the combination of amphotericin B with zero-dimensional N-CQDs to form a

84

composite probe for detecting C. albicans. In the present study, agricultural cornstalk as

85

a carbon source was used to synthesize smaller CQDs by hydrothermal carbonization

86

(200°C, 6 h). Ethanediamine was used as a nitrogen source to obtain more stable

87

N-CQDs via the hydrothermal method. Due to their superior optical properties, the

88

N-CQDs modified with water-soluble amphotericin B or alcohol-soluble amphotericin

89

B were applied to detect C. albicans. The practicability of this sensing approach was

90

validated by recovery experiments detecting C. albicans in a multi-matrix. Ultimately,

29

and drug efficacy


30.

However, no studies have


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91

the current strategy of using N-CQDs conjugated with amphotericin B to detect C.

92

albicans is regarded as an innovative bioassay.

93

RESULTS AND DISCUSSION

94

Characterization of the CQDs and N-CQDs. The size distribution and morphology of

95

the obtained CQDs from cornstalk were characterized by transmission electron

96

microscopy (TEM) and high-resolution transmission electron microscopy (HR-TEM).

97

As shown in Fig. 1A, the CQDs were nearly spherical and well dispersed. The inset of

98

Fig. 1A shows a crystalline structure with lattice spacing distances of approximately

99

0.191 nm. The size distribution of the CQDs is illustrated in the histogram (Fig. 1C),

100

and the average diameter of the CQDs was found to be approximately 2.858 nm. The

101

size of the newly synthesized CQDs was smaller than that previously reported

102

obtained N-CQDs had a size distribution from 0.5 to 4.5 nm, with a similar crystalline

103

structure, according to TEM and HR-TEM images in Fig. 1B and D.


21.

The

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104 105

Fig. 1 TEM images of (A) CQDs and (B) N-CQDs with the inset of HR-TEM and

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diameter distribution of (C) CQDs and (D) N-CQDs.

107

To identify the functional groups of CQDs, N-CQDs and N-CQDs@AmpB, fourier

108

transform infrared spectra (FTIR) were recorded. As shown in Fig. 2. The broad band at

109

3405 cm-1 in the region of 3300-3500 cm-1 was associated with hydroxyl groups. A

110

strong absorption band at 1046 cm-1 was associated with the stretching vibration of

111

alcoholic hydroxyl (C-O) or ether groups (C-O-C). A weak absorption band that

112

appeared at 1617 cm-1 was associated with the presence of C=O. Compared with that of

113

the CQDs, the FTIR spectrum of the N-CQDs revealed that the N-CQDs mainly

114

contained hydroxyl (3445 cm-1), amidogen (3251 cm-1), C-H (2843 cm-1), C=O (1639

115

cm-1), aromatic nitro (1527 cm-1), C-OH (1330 cm-1), C-N (1041 cm-1) and N-H (650

116

cm-1) functional groups or chemical bonds (Fig. 2). Thus, the presence of these groups



117

indicated that nitrogen atoms were doped into the CQDs 31.

118 119

Fig. 2 FTIR spectra of the synthesized CQDs, N-CQDs and N-CQDs@AmpB.

120

To further understand the functional groups and element states of the CQDs and

121

N-CQDs, a series of X-ray photoelectron spectra (XPS) analyses were carried out. Fig.

122

S1A shows the XPS spectrum of the CQDs, and the peaks represent the carbon (C1s),

123

oxygen (O1s) and nitrogen (N1s) signals in the spectrum. The high-resolution spectra of

124

the C1s peak (Fig. S1B) could be devolved into two peaks at approximately 284.7 eV

125

and 288.7 eV, which were attributed to C-C/C=C and C=O bonds, respectively. The

126

N1s spectra showed a peak (Fig. S1C) at 400.1 eV, which was attributed to N-H groups.

127

The high-resolution spectra of the O1s peak (Fig. S1D) displayed a peak at 531.9 eV,

128

which was ascribed to C-OH/C=O groups 32. The XPS results revealed that the surface

129

of the CQDs had hydroxyl groups and carbonyl functional groups. The functional

130

groups identified by XPS were in good agreement with those identified by FTIR

131

spectroscopy.

132

Fig. S1E shows the XPS spectrum of the N-CQDs. The XPS measurement indicated


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133

the existence of three elements in N-CQDs. Fig. S1F-H shows the high-resolution XPS

134

spectra of C1s, N1s and O1s from the N-CQDs. The C1s spectra showed three peaks at

135

285.5 eV, 285.5 eV and 288.6 eV, which were attributed to C=C/C-C, C-O and C=O

136

groups. The N1s spectra showed a peak at 400.2 eV, which was attributed to N-H

137

groups. Meanwhile, the O1s spectra showed a peak at 532 eV, which was attributed to

138

C=O groups

139

42.3% higher than that of the CQDs. Thus, it could be demonstrated that doping N in

140

CQDs was a successful operation and that the N-CQDs included diverse functional

141

groups that accounted for the higher fluorescence intensity and stability.

33.

The above data show that the nitrogen content of the N-CQDs was

142

The UV-visible absorption spectrum (UV) of the CQDs showed one characteristic

143

peak (Fig. 3A). The peak at 220 nm corresponded to the π-π* transition of the C=C

144

bond, and the peak at 281 nm corresponded to the n-π* transition of the C=O bond. The

145

CQDs emitted blue light under UV irradiation (365 nm) in aqueous solution, as shown

146

in the inset of Fig. 3A. The N-CQD solution was light brown under room light and

147

emitted intense blue light under UV irradiation (365 nm). Two weak UV absorption

148

peaks appeared at 278 nm and 340 nm (Fig. 3A). As shown in Fig. 3B, the maximum

149

excitation and emission spectra of the synthesized CQDs were recorded as 360 nm and

150

450 nm, respectively. After doping with nitrogen, the optimum excitation and emission

151

wavelengths were 381 nm and 467 nm, respectively (Fig. 3B). Compared with that of

152

the CQDs, the fluorescence intensity of the N-CQDs greatly improved, with the

153

appearance of a new UV absorption peak (340 nm). The results indicated that the



154

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groups on the surface of the CQDs had changed.

155 156

Fig. 3 (A) UV-vis absorption spectra of CQDs and N-CQDs, and the insets display the

157

images of CQDs and N-CQDs under daylight and UV irradiation. (B) Fluorescence

158

excitation and emission spectra of CQDs and N-CQDs.

159

Characterization of the N-CQDs@AmpB. It was reported that water-soluble

160

vancomycin modified with CQDs could be used to detect S. aureus

161

considering the effect of solubility on synthesizing N-CQDs@AmpB, amphotericin B

162

deoxycholic acid sodium was first selected to modify the N-CQDs. Amphotericin B was

163

easily conjugated with the N-CQDs because it contains amidogen and carboxyl groups

164

(Fig. S2). To achieve accurate detection for C. albicans, amphotericin B was modified

165

on the surfaces of N-CQDs through the condensation reaction of the amine and carboxyl

166

groups, in which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)

167

and N-Hydroxysulfosuccinimide (NHS) functioned as crosslinking agents. The FTIR

168

spectrum of N-CQDs@AmpB is also given in Fig. 2. The broad band at 3428 cm-1 in

169

the 3300-3500 cm-1 region was associated with hydroxyl bands, and the stretching

170

vibration band of C=O was identified at 1641 cm-1. The peaks at 2846 cm-1, 1546 cm-1,


24.

Similarly,

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171

1375 cm-1 and 1041 cm-1 corresponded to stretching vibrations C-H, -NO2, C-OH, and

172

C-N, respectively. Moreover, the peak at 605 cm-1 indicated the existence of the N-H

173

group. A sharp peak at 2945 cm-1 was associated with the stretching vibration of

174

alcoholic CO-NH. Signals of new amide bond functional groups appeared in the FTIR

175

spectrum of the composite probe, indicating that amphotericin B and N-CQDs were

176

bound together via bonds rather than physical adsorption. As shown in Fig. S3A, the

177

fluorescence intensity of N-CQD@AmpB showed minimal variation in the fluorescence

178

spectra compared with that of the N-CQDs themselves. The UV absorption spectra of

179

N-CQDs, N-CQD@AmpB and amphotericin B were also obtained, as shown in Fig.

180

S3B. The N-CQDs showed an absorption peak at 278 nm. Amphotericin B showed

181

three absorption peaks at 282 nm, 322 nm and 384 nm, while N-CQDs@AmpB

182

exhibited three higher-intensity absorption peaks associated with N-CQDs and

183

amphotericin B. The above evidence showed that amphotericin B had minimal effect on

184

the properties of N-CQDs when conjugated with them. In addition, varying pH values,

185

temperatures and concentrations of NaCl were introduced to explore their effects on

186

N-CQDs and N-CQDs@AmpB (Fig. S4, S5, and S6), the results demonstrated that the

187

bond between N-CQDs and amphotericin B was not greatly affected by those factors.

188

Establishing the assay and its mechanism. As is known, amphotericin B can

189

specifically combine with ergosterol of the fungus cell membrane

190

could be indirectly linked with C. albicans (Fig. 4). Based on this mechanism, an assay

191

was designed to be applied for detecting fungus by N-CQDs@AmpB. Although the


25,

so the N-CQDs


192

attachment of amphotericin B to other nanomaterials such as carbon nanotubes was

193

reported previously, the synthesis and application of amphotericin B linked with carbon

194

nanotubes were both different from those used for the composite probe in this paper. As

195

shown in Fig. 5A, when C. albicans was added to N-CQDs@AmpB, a dramatic

196

fluorescence increase was observed, indicating their availability for detecting C.

197

albicans. However, when the same amount of C. albicans was added to amphotericin B,

198

there was minimal fluorescence (Fig. S7). The results demonstrated that the

199

fluorescence was derived from N-CQDs rather than amphotericin B. Then, serving as

200

the control experiment, the N-CQDs without modifications were mixed with C. albicans

201

in phosphate buffered saline (PBS) (20 mM, pH 7.4). As shown in Fig. 5B, the

202

fluorescence intensity of N-CQDs varied slightly with varying concentrations of added

203

C. albicans. The results indicated that there were no direct interactions between C.

204

albicans and N-CQDs without amphotericin B. Thus, N-CQDs@AmpB rather than

205

N-CQDs played the role as a probe for C. albicans.

206 207

Fig. 4 Schematic illustration for detecting C. albicans.


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208 209

Fig. 5 (A) Fluorescence spectra of N-CQDs@AmpB in the absence (black), presence

210

(red) of C. albicans (1.0 x 108 cfu/mL). (B) Fluorescence spectra of N-CQDs in the

211

presence of different concentrations of C. albicans.

212

To further demonstrate the interactions between N-CQD@AmpB and C. albicans,

213

many related experiments were designed. First, C. albicans was diluted 1000 (Fig. S8A,

214

C, and E) and 10000 (Fig. S8B, D, and F) times and plated for counting. In Fig. S8A

215

and B, the C. albicans samples without added N-CQDs or N-CQDs@AmpB served as

216

controls, exhibiting normal growth. Similarly, the amounts of fungal colonies slightly

217

decreased when C. albicans was incubated with N-CQDs at 37°C for 24 h (Fig. S8C

218

and D), indicating that the unmodified N-CQDs barely interacted with C. albicans and

219

had low toxicity. Compared with those shown in Fig. S8A and B, a distinct decrease in

220

the amount of fungal colonies was observed when C. albicans was incubated with

221

N-CQDs@AmpB (Fig. S8E and F). This result showed that N-CQDs@AmpB could

222

restrain the C. albicans population to some extent. Taken together, these results directly

223

demonstrated that N-CQDs@AmpB could interact with C. albicans on the cell surfaces



224

through amphotericin B.

225

Optimization of detection conditions. Two main factors possibly affected the

226

detection of fungus. One main factor was the concentration of amphotericin B, so

227

different concentrations of amphotericin B were introduced while synthesizing

228

N-CQDs@AmpB. As shown in Fig. S9A, 1 mg/mL of amphotericin B was optimal.

229

Another factor was incubation time. Likewise, varying times (5 min, 15 min, 30 min, 50

230

min, 70 min, 90 min and 100 min) for the reaction between N-CQDs@AmpB and C.

231

albicans were studied. However, the result was very interesting, and the fluorescence of

232

N-CQDs@AmpB remained at the same level as the reaction time increased in Fig. S9B.

233

This result is attributed to the fact that N-CQDs@AmpB could combine with C.

234

albicans in a short time. Thus, 5 min served as the optimal reaction time. Eventually, 1

235

mg/mL of amphotericin B and 5 min of incubation time were identified as the optimal

236

conditions during the detection of C. albicans.

237

Detection of C. albicans by using N-CQDs@AmpB. The standard curve was

238

established by testing various concentrations of C. albicans (2.60 x 105 - 1.99 x 108

239

cfu/mL). The results (Fig. 6A and B) indicated that a series of different concentrations

240

of C. albicans labeled with N-CQDs@AmpB showed a great linear range of 2.60 x 105

241

– 1.99 x 108 cfu/mL with a correlation coefficient of 0.9912. The fluorescence intensity

242

of C. albicans labeled with N-CQDs@AmpB linearly increased with increasing

243

concentrations of fungal cells, with a limit of detection (LOD; at a S/N ratio of 3) of

244

approximately 1124 cfu/mL 34. Considering the high cost of water-soluble amphotericin


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245

B and the large-scale application of the new detection method, water-soluble

246

amphotericin B was replaced by alcohol-soluble amphotericin B to synthesize

247

N-CQDs@AmpB (alcohol-soluble). The resultant fluorescent probe showed a greater

248

linear range of 4.30 x 105 – 1.86 x 108 cfu/mL and a correlation coefficient of 0.9931

249

(Fig. S10). The fluorescence intensity of C. albicans labeled with N-CQDs@AmpB

250

(alcohol-soluble) linearly increased with increasing concentrations of fungal cells, with

251

a LOD (at a S/N ratio of 3) of approximately 2485 cfu/mL. Although the detection limit

252

of N-CQDs@AmpB (alcohol-soluble) was slightly high, it was still necessary for

253

detecting fungus considering the cost. The results provided the possibility for

254

N-CQDs@AmpB to detect other fungi that are sensitive to amphotericin B. This new

255

approach broadens avenues for determining the presence and amounts of fungi.

256 257

Fig. 6 (A) Fluorescence spectra at 355 nm excitation of C. albicans (2.6 x 105 - 1.99 x

258

108 cfu/mL) labeled with N-CQDs@AmpB. (B) Plot of fluorescence intensity at 355

259

nm excitation with respect to C. albicans concentration.

260

Specificity of N-CQDs@AmpB. Regarding the specificity of N-CQDs@AmpB, three

261

different types of microorganisms, including C. albicans, Escherichia coli and



262

Trichosporon fermentans, were separately introduced into the same amount of

263

N-CQDs@AmpB, and the fluorescence intensity was then measured. As shown in Fig.

264

7, a dramatic increase in fluorescence was observed for N-CQDs@AmpB with

265

additions of C. albicans, indicating the sensitive response of N-CQDs@AmpB to C.

266

albicans. In contrast, there was no obvious fluorescence increase observed by adding E.

267

coli or T. fermentans into the N-CQDs@AmpB solution, revealing the satisfactory

268

selectivity of N-CQDs@AmpB for C. albicans.

269 270

Fig. 7 Fluorescence emission spectra at 355 nm excitation of C. albicans (1 x 108

271

cfu/mL) (blue), E. coli (1 x 108 cfu/mL) (red) and T. fermentans (1 x 108 cfu/mL)

272

(black) labeled with N-CQDs@AmpB.

273

Analysis of C. albicans in beef sausage. To verify its applicability, this approach was

274

employed to detect C. albicans in sabaurauds agar (SAB) media containing beef

275

sausage for exploring the recovery of the organism. First, beef sausage was sterilized by

276

autoclaving to remove other microorganisms. Then, C. albicans was incubated with


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277

media containing beef sausage at 37°C for different times to obtain different

278

concentrations of fungal suspension, and the amount of C. albicans was calculated by

279

the standard curve and regression equation. It was important that the recoveries obtained

280

were both above 90% in all cases, as listed in Table 1 and Table 2, suggesting a

281

generally satisfactory recovery and RSD 24. We also set up a control group to measure

282

the amount of microorganisms during the same time by plate count to guarantee the

283

accuracy of the data. These data suggested that using N-CQDs@AmpB (water-soluble

284

or alcohol-soluble) as a probe to detect C. albicans in a real sample was available.

285

Table 1: Recoveries of C. albicans growing in beef sausage directly detected by

286

N-CQDs@AmpB (water-soluble) (n=6) Sample

Control (cfu/mL)

Measure (cfu/mL)

Recovery (%)

RSD (%)

1

5.48 x 106

5.36 x 106

97.81

0.49

2

6.28 x 106

5.69 x 106

90.61

0.81

3

7.36 x 107

6.96 x 107

94.57

0.61

4

8.75 x 107

9.06 x 107

103.54

1.02

287

Table 2: Recoveries of C. albicans growing in beef sausage directly detected by

288

N-CQDs@AmpB (alcohol-soluble) (n=6) Sample

Control (cfu/mL)

Measure (cfu/mL)

Recovery (%)

RSD (%)

1

3.65x106

3.83x106

104.91

1.29

2

3.96x106

3.78x106

95.45

1.31

3

4.47x107

4.59x107

102.68

0.86

4

4.68x107

4.63x107

98.93

0.37

289

CONCLUSIONS

290

In summary, we took advantage of fluorescent N-CQDs originating from cornstalk to



291

detect pathogenic fungi. To the best of our knowledge, this is the first report to detect C.

292

albicans by N-CQDs modified with amphotericin B. Specifically, the connection of

293

N-CQDs@AmpB with C. albicans occurred on the surfaces of fungi based on

294

ligand-receptor interactions, thereby leading to increased fluorescence. The linear range

295

of 2.60 x 105–1.99 x 108 cfu/mL for C. albicans was acceptable, and the N-CQDs

296

modified with amphotericin B could efficiently detect C. albicans with high sensitivity

297

and selectivity. Considering the economy of the process, alcohol-soluble amphotericin

298

B could reduce the cost of the assay, but its LOD was higher than that of water-soluble

299

amphotericin B. An approach using N-CQDs modified with amphotericin B to detect

300

target fungi in complex samples was developed. This detection method is expected to

301

find application in other areas as well.

302

EXPERIMENTAL SECTION

303

Materials. Cornstalk and beef sausage were bought from a local market in Jilin.

304

Ethanediamine was obtained from Tianjin Guangfu Chemical Research Institute. E.

305

coli, T. fermentans and C. albicans were purchased from Guangdong Microbial Culture

306

Center. Tryptone, peptone, yeast extract, anhydrous glucose, agar and amphotericin B

307

(water-soluble and alcohol-soluble) were obtained from Beijing Coolaber Science &

308

Technology Co., Ltd. NHS and EDC were purchased from Beijing Hanlongda

309

Technology Development Co., Ltd. All solvents were analytical grade and used without

310

further purification. Ultrapure water (18.2 M Ώ cm-1) purified by a Merck Millipore

311

system was used in the experiments.


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312

Instrumentation. The size and morphology were studied by HRTEM (Hitachi, Japan)

313

on a Hitachi H-800 microscope with an accelerating voltage of 200 kV. UV (UV-2550,

314

Shimadzu, Japan) spectra were obtained using a Shimadzu computer-controlled double

315

beam UV-vis spectrophotometer. FTIR (IRAffinity-1 Shimadzu, Japan) spectra were

316

recorded with an FTIR spectrometer (32 scans). The fluorescence spectra were recorded

317

with a single-beam spectra fluorophotometer (RF-5301PC, Shimadzu, Japan). XPS

318

were recorded on a PHI Quantera II system (Ulvac-PHI, INC, Japan). Cell images were

319

recorded using a fluorescence microscope (DFM55, Ningbo, China).

320

Synthesis of CQDs from cornstalk. CQDs were synthesized by hydrothermal

321

carbonization. First, the cornstalk was cut into small pieces and dried in sunlight for

322

several days. Next, the shredded cornstalk was weighed 0.3 g and mixed with distilled

323

water (30 mL) in a glass bottle. Afterwards, the mixture was ultrasonicated for 10 min,

324

and then, the mixture was placed into a Teflon-lined autoclave at 200°C for 6 h. After

325

cooling to room temperature, the dispersed carbon solution was centrifuged at 12,000

326

rpm for 15 minutes at room temperature. Finally, the supernatant liquid was collected in

327

a new glass bottle, passed through a 0.22 µm micron filter and dialyzed against

328

deionized water through a dialysis membrane (1000 Da) for 2-3 days. The obtained

329

CQDs were stored at room temperature.

330

Synthesis of N-CQDs. Ten milliliters of CQDs and 1 mL ethanediamine were placed in

331

a bottle and then stirred for 10 min. Next, the mixture was transferred into a

332

Teflon-lined autoclave and heated at 180°C for 5 h in an oven


35.

Ultimately, after


Page 20 of 26

333

cooling to room temperature, the mixture was centrifuged at 13,000 rpm for 10 min.

334

The supernatant was passed through a 0.22 µm micron filter and stored in a new glass

335

bottle at room temperature.

336

Synthesis of N-CQDs@AmpB. In brief, 50 mg each of fresh EDC and NHS was

337

successfully introduced into 5 mL N-CQDs for activating the carboxyl groups on their

338

surface, and the reaction was allowed to rotate for 4 h at room temperature. Next, 7 mg

339

amphotericin B (water-soluble) was dissolved in 2 mL PBS (20 mM, pH 7.4), forming a

340

transparent solution. Immediately, 2 mL of amphotericin B solution was added to this

341

N-CQD solution and further gently stirred for 6 h at room temperature. Ultimately, 1000

342

Da of dialysis membrane was applied to remove free amphotericin B and N-CQDs

343

N-CQDs@AmpB (alcohol-soluble) could be synthesized by the same method except

344

that 7 mg amphotericin B (alcohol-soluble) was dissolved in 2 mL ethanol.

345

Microorganism growth and assays. Microorganism culturing and assays were

346

performed in a sterile clean room. C. albicans and E. coli were cultured in SAB and

347

Luria-Bertani (LB) media at 37°C for 12 h, respectively. T. fermentans grew in sterile

348

yeast extract peptone dextrose (YEPD) media in an incubator shaker at 30°C for 24 h.

349

Then, 1 mL from each cultured medium was centrifuged at 10000 rpm for 10 min to

350

remove the supernatant and washed 3 times with PBS and then suspended in 1 mL PBS.

351

Afterwards, the microorganism concentration was adjusted to an appropriate level by

352

measuring the optical density (OD) at 600 nm. By plating C. albicans, E. coli and T.

353

fermentans on SAB, LB and YEPD plates, respectively, the amounts of bacteria or


24.

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354

fungi per milliliter could be acquired by counting related colony forming units (cfu)

355

after incubation. For detection, microorganism samples were diluted into various

356

concentrations using PBS buffer. Then, 1 mL N-CQDs@AmpB was mixed with 1 mL

357

each sample described here and incubated for 5 min, the fluorescence variations of

358

N-CQDs@AmpB were further recorded by fluorescence spectrometry with an

359

excitation wavelength of 355 nm.

360

Detecting target fungus in beef sausage. To investigate the practical applicability of

361

N-CQDs@AmpB to a real sample, C. albicans was cultured in SAB media containing a

362

patch of sterile beef sausage at different times to obtain various concentrations of fungal

363

suspension. Then, the prepared samples were used for fungal detection with a general

364

procedure. In addition, the plate count method for assaying the prepared samples served

365

as the controls.

366

AUTHOR INFORMATION

367

Corresponding authors

368

E-mail: [email protected] (Yu. D.Y.); [email protected] (Qiao N.).

369

CONFLICTS OF INTEREST

370

The authors declare that the research was conducted in the absence of any commercial

371

or financial relationships that could be construed as a potential conflict of interest.

372

ACKNOWLEDGEMENTS

373

This work was supported by grants from the National Natural Science Foundation of

374

China (31470787, 21708003) and Science and Technology Research Project of Jilin



375

Province, China (20170519015JH).

376

SUPPLEMENTARY INFORMATION

377

The Supporting Information is available free of charge on the ACS Publications

378

website.

379

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380

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For Table of Contents Use Only

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Fluorimetric detection of Candida albicans using cornstalk N-carbon

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