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Novel Pyrimidine tagged Silver Nanoparticles based Fluorescent Immunoassay for detection of Pseudomonas aeruginosa Ellairaja Sundaram, krithiga N, Sarkaraisamy Ponmariappan, and Vairathevar Sivasamy Vasantha J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04790 • Publication Date (Web): 05 Feb 2017 Downloaded from http://pubs.acs.org on February 6, 2017

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Journal of Agricultural and Food Chemistry

Novel Pyrimidine tagged Silver Nanoparticles based Fluorescent Immunoassay for detection of Pseudomonas aeruginosa

1 2 3 4

Sundaram Ellairaja a* Narayanaswamy Krithiga b*, Sarkaraisamy Ponmariappan Vairathevar Sivasamy Vasantha *†

5

a

6

Madurai – 625 021, Tamilnadu, India.

7

b

8

– 625 021, Tamilnadu, India.

9

c

10

c

* and

*†Department of Natural Products Chemistry, School of Chemistry, Madurai Kamaraj University, * Department of Plant Biotechnology, School of Biotechnology , Madurai Kamaraj University, Madurai

* Biotechnology Division, Defence Research Development & Establishment, Jhansi Road, Gwalior

474002, Madhya Pradesh, India.

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

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Abstract

31

A simple pyrimidine-based fluorescent probe (R)-4-(anthracen-9-yl) -6- (naphthalen-1-yl)-1,6-

32

dihydropyrimidine-2-amine (ANDPA) was synthesized through the greener one pot reaction and

33

characterized by IR, NMR and ESI-Mass. Glucose stabilized silver nanoparticles (Glu-AgNPs)

34

were also synthesized and characterized using UV, IR, XRD, SEM & TEM. When ANDPA was

35

tagged with Glu-AgNPs, fluorescent intensity of ANDPA decreased drastically. When the

36

monoclonal antibody (Ab) [immunoglobulin G (IgG)] of Pseudomonas aeruginosa (PA) was

37

attached with ANDPA/Glu-AgNPs, the original intensity of the probe was recovered with

38

minimal enhancement at 446 nm. On further attachment of PA with ANDPA/Glu-AgNPs/PA,

39

the fluorescence intensity of the probe was enhanced obviously at 446 nm with red shift. This

40

phenomenon was further supported by SEM and TEM. The linear range of detection is from 8 to

41

10-1 CFU/mL and LOD is 1.5 CFU/mL. The immunosensor was successfully demonstrated to

42

Pseudomonas aeruginosa in water, soil and food products like milk, sugarcane and orange

43

juices.

44 45 46 47 48 49 50 51 52 53 54 55 56 57

Keywords: Pyrimidine, Fluorescence, Aggregation, Pseudomonas aeruginosa, Nanoparticles, Immunosensor. 2 ACS Paragon Plus Environment

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■ Introduction

59

Amidst Pseudomonadacae family, Pseudomonas aeruginosa has been pointed out as an

60

opportunistic gram negative human pathogen that causes acute diseases in human as well as

61

animals.1-2 Pseudomonas aeruginosa pathogen is the fourth most commonly-isolated nosocomial

62

pathogen accounting for 10.1 percent of all hospital-acquired. Several serious health issues by

63

this pathogen are sepsis, inflammatory, fester otitis, pneumonia and cystic fibrosis. And also it is

64

consider as a primary cause of morbidity and mortality in patients with cystic fibrosis. In recent

65

decades, Pseudomonas aeruginosa based infections are very firm to extirpate and the current

66

diagnostic tools are facing sundry of challenges. Pseudomonas aeruginosa finds numerous

67

reservoirs such as disinfectants, respiratory equipment, food, sinks, taps, and mops. This

68

organism is often reintroduced into the hospital environment on fruits, plants, vegetables, as well

69

by visitors and patients. Spread occurs from patient to patient on the hands of hospital personnel,

70

by direct patient contact with contaminated reservoirs, and by the ingestion of contaminated

71

foods and water.3 Moreover, Pseudomonas aeruginosa has showed an immense potential to

72

develop resistance against antibiotic as is evident from the fact that its genome contains the

73

largest resistance for more than 50 resistance genes. So, the rapid and sensitive detection of

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Pseudomonas aeruginosa is extremely important in biological research and medical diagnosis.

75

In concern with the sensitivity, usage, time, selectivity etc., so many analytical protocols

76

such as polymerase chain reaction (PCR), microarrays,4 quartz crystal microbalance resonators

77

(QCM),5 fluorescent

78

ferromagnetic

79

glyconanoparticles,8 light-addressable potentiometric detection,9 amperometric detection of

80

enzymatic reaction products,10 diffraction-based cell detection,11 and nanowire-based detection 12

81

are continually being developed for the recent decades for various pathogens.13 By combining

82

the speed and sensitivity, recently multiplexed flow cytometry method was reported for detecting

83

the human pathogens.14

bioconjugate

nanoparticles,7

silica

nanoparticles,6

carbohydrate-mediated

monoclonal

cell

antibody-coupled

recognition

using

gold

84

Besides, a highly specific and sensitive Genome Exponential Amplification Reaction

85

(GEAR) assay for the detection of E. coli has been designed.15 After a keen reviewing of the

86

recent detection protocols, the microarray based protocol can detect a minimum of 6000 cells of

87

E. coli from a suspension of 5x106 cells/ mL. Similarly, A silicon chip-based light-addressable 3 ACS Paragon Plus Environment


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potentiometric biosensor can detect 119 Salmonella cells from suspension of 106 cells/mL.9

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Though the existence of so many sophisticated analytical tools, still now a challenge is there to

90

develop a highly sensitive, speedy, very simple and cost effective pathogen based sensors,

91

because all these methods require signal amplification or enrichment of the target bacteria in the

92

sample or expression of fluorescent protein markers and antibodies in the cells. As a

93

consequence, these methods tend to include additional steps and time-consuming assay

94

procedures.16 To address these short comings, recently fluorimetric technique based

95

immunoassay is offering a high accuracy, speed, simplicity and cost effectiveness. In past years,

96

the labeling of growing pathogens with fluorescent tags during the bacterial culture had been

97

played as a remarkable part in fluorescent microscope based bacterial detection.17 Now, its

98

application is extended and widely spreads in bacterial/pathogen detections also.18-21 A

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fluorescence-based sensor system can detect 480 Pseudomonas aeruginosa cells present in a

100

suspension of 2.4 x 105cells/ mL.22

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Recently, the interactions of fluorophores with metallic nanoparticles (Nps) have gained

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considerable interest due to their increased use in the detection of inorganic metal ions, viruses,

103

DNA and proteins.23 Owing to its high surface to volume ratio, it can be easily labeled with large

104

amounts of different molecules and their physical properties are also chemically tailorable.24-25

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The conjugation of fluorescent molecules on the nano surface could be achieved based on the

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intermolecular attractions like covalent/non-covalent bonding, chemisorptions etc. During

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interaction, there is an enhancement or quenching phenomenon could occur.26-27 The

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fluorescence response could be enhanced or quenched when the fluorophore is localized near a

109

metal surface, which is purely related to the shape and size of the metal particle, and the distance

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between the dye and the metal surface.28-29 Moreover, the nanoparticles possess high surface to

111

volume ratio which will be useful for the increasing the number of antibody or antigens etc. on

112

its surface.30 Hence, various nanoparticle – fluorescent tag based immunoassay have been

113

reported for pathogen detection 31-34 .

114

A simple immunosensor based on the fluorescent labeled nano particles such as

115

CdSe/ZnS@SiO2 had been developed for the detection of Salmonella Typhimurium bacterium.35

116

Ruthenium Bipyridine-doped silica nanoparticles and multifunctional graphene magnetic

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nanosheets decorated with chitosan as fluorescent tag for bacterial detection had already been

118

reported.36-38 A fluorescent molecule labeled nanomagentic beads based an in-stiu immuno 4 ACS Paragon Plus Environment

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protocol for food borne pathogen has developed. The above sensor showed a multiplex detection

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of three food pathogens namely Escherichia coli O157:H7, Salmonella typhimurium, and

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Listeria monocytogenes in buffer in a simultaneous manner.39 Recently, a label free biosensor

122

has developed through laser beam technique for the detection of Escherichia Coli. But very rare

123

fluorescent and electrochemical biosensors were reported for Pseudomonas aeruginosa

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bacterium like G. Kaur et al also reported a fluorescent organic nanoparticle based biosensor for

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the bacterium Pseudomonas aeruginosa with the very good selectivity and sensitivity recently40

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and very recently our group has designed simple electrochemical immunoassay based on pectin –

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gold nanocomposite platform for the Pseudomonas aeruginosa detection within in the linear

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range of 101 to 107 CFU/mL and LOD is 9X102 CFU/ml.41 So, the development of immunoassay

129

protocol for Pseudomonas aeruginosa is being increased while comparing with other clinical

130

diagnostic protocols.

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Pyrimidine molecule is one of the important heterocycles and pharmacophor in our

132

biological system. There were so many pyrimidine based organic molecules were synthesized

133

through various methodology which involving some crucial and multi steps and also longer

134

reaction time. In order to overcome these demerits, we have synthesized a simple and very new

135

pyridimine based fluorescent molecule (ANDPA) via sonochemical technique within 35 mins at

136

50-600C with good fluorescent properties with a maximum yield (1.89g) of 98% at 30mins. 42-44 In this report, we have synthesized glucose stabilized AgNPs (Glu-AgNPs) and tagged

137 138

with

our

synthesized

ANDPA

molecules.

Then,

the

monocolonal

antibody

(Ab)

139

(Immunoglobulin G (IgG)) of Pseudomonas aeruginosa pathogens was attached with

140

ANDPA/Glu-AgNPs and applied for the detection of the target pathogen in the presence of

141

other interferences and finally applied detection of Pseudomonas aeruginosa in water,

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agriculture samples soil and food items like milk, sugarcane and orange juices successfully. To

143

the best of our knowledge, this is the first report for the green synthesis of pyrimidine-based

144

fluorescent probe (ANDPA) and second report about fluorescent immunosensor for the detection

145

of Pseudomonas aeruginosa.

146 147

■ Materials and Methods

148

1-Acetyl Naphthalene, Anthracene 9- carbaldehyde, Guaninidine hydrochloride, NaOH pellets

149

disodium hydrogen phosphate, monosodium dihydrogen phosphate and bovine serum albumin 5 ACS Paragon Plus Environment


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(BSA) and HPLC grade ethanol were purchased from Sigma Aldrich, USA via dealers;

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Ponmanichem Glass Agencies, Madurai, Tamilnadu, India and used as such. Pseudomonas

152

aeruginosa Monoclonal Antibody IgG [B11] is purchased from ThermoFisher Scientic

153

Company, USA via dealers; Invitrogen BioServices Pvt Ltd.

154

Bacterial strains like Salmonella paratyphi A (S. paratyphi A) MTCC 735, Klebsiella

155

pneumoniae (K. pneumoniae) MTCC 432, Staphylococcus Aureus (MTCC96), Escherichia Coli

156

(MTCC 448), Pseudomonas Stutteri (MTCC 101) and Pseudomonas aeruginosa (MTCC 2534)

157

were originally obtained from Microbial Type Culture Collection Centre, Institute of Microbial

158

Technology, Chandigarh, India. Water samples were collected from different districts Madurai,

159

Chennai, Virudhunagar, Dindigul and Salem, Tamilnadu, India. Agricultural soils were collected

160

from Usilampati, Madurai district, Tamilnadu, India. And food items like milk, sugarcane and

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orange were purchased from local shops, Madurai. All the reagents used for the experiments

162

were prepared in doubly distilled water. 1H &

163

MHz spectrometer in using CDCl3 and DMSO-d6 as solvents containing a trace quantity of

164

tetramethylsilane (TMS) as internal standard. The chemical shifts are reported in ppm at 25 °C.

165

Mass spectra and UV spectra were recorded using ESI ionization in MS-LCMS mass

166

spectrometer and SHIMADZU single beam UV−vis spectrophotometer, respectively.

167

Fluorescent measurements were done using Cary Eclipse spectrophotometer having a 450 W

168

xenon lamp. The 5 nm and 2.5 nm were maintained as excitation and emission slit widths,

169

respectively throughout the experiments.

Bangalore, Karnataka, India.

13

C NMR analysis was done using Bruker- 300

170 171

One pot green Synthesis of (R)-4-(anthracen-9-yl) -6- (naphthalen-1-yl)-1, 6-dihydro

172

Pyrimidine-2-amine (ANDPA):

173

For the very first time, we have synthesized a simple pyrimidine based fluorescent probe through

174

ultrasonication method. Briefly, one equivalent of acetyl naphthalene (1g, 0.00580 mol), one

175

equivalent of antharacene aldehyde (1.2g, 0.0058 mol) and one equivalent of guanidine

176

hydrochloride (0.5g, 0.0058 mol) were dissolved in ethanol with an excess of 0.1M NaOH (5

177

mL) and then subjected to ultrasound irradiation for 30 mins. The initial pale yellow color was

178

changed to dark yellow at the end of the reaction. The completion of the reaction was monitored

179

by TLC and confirmed by observing the appearance of the single yellow spot. Then, the solution

180

was poured into ice-cold water, filtered and washed thrice with absolute ethano1. The yield was 6 ACS Paragon Plus Environment

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found to be 98% (1.89g) and the product was isolated by using diethyl ether and water mixture.

182

Then, the ether layer was separated and evaporated and finally, the yellow color solid obtained

183

was recrystallized using absolute hot ethanol to obtain the desired product (Figure 1a). The

184

melting point (uncorrected) of the synthesized ANDPA was found to be 1300C.

185

The NMR data for the synthesized imine are given below. 1H NMR (300 MHz, CDCl3, 298): δ =

186

8.49-8.34 (m, 2H), 8.02 (s, J=8.3Hz, 2H), 7.70-7.75 (m, 3 H), 7.60-7.21 (m, 12 H), 6.93(s, 1H),

187

5.05 (d, J=2.5Hz, 1H) ppm.13C NMR (300 MHz, DMSO, 298 K): δ = 152.82, 142.15, 137.75,

188

134.36, 132.65, 130.41, 128.23, 127.08, 126.99, 126.61, 125.53, 124.54, 124.50, 124.38, 123.97,

189

101.94, 49.84 C28H21N3 (calculated mass 399.17): observed mass [M + H]+ =400.24 [Figure S1,

190

S2 &S3].

191

Bio-green synthesis of glucose stabilized silver nanoparticles (Glu-AgNPs):

192

We have followed the previous report and just modified the procedure for the synthesis of

193

AgNPs.45 Briefly, 0.45 g of glucose was dissolved in 5 mL of 0.1 M NaOH and gently heated

194

with string. Then, 9 ml of the above solution was added to 1mL of 1 mM silver nitrate solution

195

with constant stirring and kept it for 20 mins in dark at room temperature. The formation of

196

silver nanoparticles was confirmed through the color change of the solution from colorless to

197

brownish yellow and also appearance of a SPR band appeared at 400 nm in UV-Visible

198

spectroscopy. The Glu-AgNPs were settled down through repeated centrifugation for 30 mins at

199

28672 G-force and then washed with distilled water until to get no red color in the presence of

200

phenol in sulfuric acid medium (which is the characteristic color test for glucose molecule). The

201

Glu-AgNPs were collected and re-dispersed in deionized water for characterization. The glucose

202

solution used could act as both reducing as well as capping agent (Figure 1b).

203

(Figure 1.)

204

Designing of immunosensor for the detection of Pseudomonas aeruginosa pathogen:

205

The Glu-AgNps were stored at light free atmosphere, whereas IgG antibody, PA and other

206

pathogens were stored at -4oC and ANDPA was stored at room temperature. When 1mL of Glu-

207

AgNPs [6 %( v/v)] was added with 2mL of ANDPA (5µM) and then it was kept at room

208

temperature in dark chamber for 10 mins and the initial blue fluorescence of the probe was 7 ACS Paragon Plus Environment


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diminished. And then 20µL of Ab (0.36µg/mL) was mixed thoroughly with the above solution

210

and incubated at -4oC for 25 mins. The original color of the probe was gradually regained.

211

Finally, 40µL of PA (10-7 µg/mL) was added to the Glu-AgNPs/ANDPA tagged Ab and

212

incubated at -4oC 25 for mins and the color of the probe has been changed to green fluorescence.

213

All those experiments were carried out in phosphate buffer (PBS, pH=7.0) medium.

214

Detection of Pseudomonas aeruginosa in Food and Agricultural samples:

215

Pseudomonas aeruginosa is mostly found in soils, lakes, rivers and fresh fruits, vegetables, etc.

216

Specifically, it was often found in swimming pools, spas, whirlpools and hot tubs since the

217

presence of high temperatures which is more appropriate condition for its growing. Meanwhile,

218

Pseudomonas strains are responsible for the milk contamination other dairy products, due to the

219

production of extracellular proteinases.46

220

To test the feasibility of the sensor, the proposed immunosensor was applied for detection of

221

Pseudomonas aeruginosa in water, soil and three different food items: cow milk, sugarcane and

222

orange juices. Water samples were collected from a river, lake and ponds of five different

223

districts in Tamilnadu. The other real samples like fresh juices were collected from orange

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fruit and sugarcane stem and cow milk (Aavin Company) was purchased from local shop,

225

Madurai. The orange fruit and sugarcane stem were crushed and the juices were filtrated

226

through Whatman (40) filter paper. Uninoculated food matrices were used as controls and then

227

Pseudomonas aeruginosa bacteria was artificially inoculated into the corresponding sample

228

medium. For testing, 9 mL of food matrices were inoculated with 1 mL of Pseudomonas

229

aeruginosa strain (24hrs old culture) and incubated at room temperature for 2 hrs. Bacteria

230

concentrations used were 265, 122, 60, 31 and 8 CFU/ mL from the dilution factor of 10-5 to 10-9.

231

From the above each sample, 40 µL of the bacterial solution was used for emission test using

232

Ab/Glu-AgNPs/ANDPA and immunoassays were performed in triplicate. The colony counting

233

method was also carried in the same experimental conditions for comparison.

234

For soil samples, 1g of testing soil was added to 10 ml of sterile saline (the stock) and shaken

235

vigorously for at least 1 minute. The dilute was then sediment for a short period. Sterile dilution

236

blanks were marked sequentially starting from stock and10-1 to 10-9. For plate count method,

237

freshly prepared plates of LB (Luria Bertani) and Cetrimide agar plates were taken. 0.1 mL of 8 ACS Paragon Plus Environment

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the serial diluted above samples were taken spread over the plates and incubated at 37 ° C for 24

239

hrs. This Cetrimide agar plates specifically allows Pseudomonas aeruginosa to grow on the plate

240

in presence of other interfering microorganisms.

241 242

■ Result and Discussion

243

Spectrochemical Property of ANDPA:

244

The photophysical properties of ANDPA were test using UV-vis and fluorescent spectroscopic

245

techniques. Firstly, ANDPA has exhibited two major absorption peaks at 260 and 372 nm, which

246

are mainly due to the n- π* and π-π* transition, respectively of ANDPA (Figure 2a). Then, the

247

emission spectra of 5M ANDPA showed three intense characteristic peaks of antharacene

248

moiety at 402, 424 and 446 nm (Figure 2b). (Figure 2.)

249 250

Effect of pH on fluorescent property of ANDPA

251

While adjusting the pH from 2 to 13, there was a remarkable quenching and spectral shift of

252

ANDPA (Figure 3). Synthesized ANDPA could exist as two resonance form in acidic and basic

253

medium. Up to pH 7, a slight decrease in fluorescent intensity was observed at 402, 424 and 446

254

nm as a result of complete protonated fluorescent moiety formation through the ICT process.

255

Whereas at above pH=7, ANDPA exhibited a single characteristic peak at 462 nm with weak

256

fluorescent and spectral shift towards longer wavelength (red shift) due to deprotonation of

257

ANDPA. On concerning the biomolecules, the neutral medium (pH 7) has been maintained for

258

our whole studies (Figure 3.).

259

Characterization of Glu-Ag nanoparticles:

260

Among all nanoparticles, silver and gold nanoparticles are considered promising as they interact

261

with incident light more efficiently than any other metal nanoparticles in the visible light range

262

due to a strong surface plasmon resonance and also these nanoparticles can easily attached

263

through lipopolysaccharide (LPS), lipoteichoic acid (LTA), protein, and phospholipids on the

264

bacterial cell surface.41 In the current work, Glu-AgNPs were synthesized and characterized 9 ACS Paragon Plus Environment


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265

through the absorption studies by observing their corresponding surface plasma resonance (SPR)

266

peak at 409 nm under optimized condition (Figure 4a). It was also confirmed by XRD analysis

267

(Figure 4b). The sharp peaks at 2θ values of 35.40°, 54.31° and 74.96° correspond to (1 1 1), (1

268

4 2), and (3 1 3) planes of silver, respectively and also the presence of crystalline pattern in Glu-

269

AgNPs. All the peaks in XRD pattern were indexed as face-centered cubic structure of silver as

270

per available literature.47-48 Using Scherrer equation, the particle size is calculated as 10 nm.

Dp = 0.94λ / β1/2COSθ

271 272

Where Dp = Average Crystallite size,

273

β

= Line broadening in radians.

274

θ = Bragg angle.

275

λ = X-ray wavelength

276 277

Interaction of ANDPA with Glu-AgNPs:

278

The tagging of ANDPA with the Glu-AgNPs was confirmed through IR spectra and UV-Visible

279

absorption spectra. In ANDPA IR spectra, the primary -NH2 stretching frequency was observed

280

at 3389 &3452 cm-1 and secondary –NH amine stretching was observed at 1626 cm-1. Whereas

281

the stretching frequencies of -C=C stretching were observed at 1369, 1446, 1553 and1566 cm-1.

282

The peaks at 2296, 2972 and 3047 cm-1 indicate the presence of –CH stretching and the peaks in

283

the region of 638-1018 cm-1 correspond to the =C-H stretching. After binding with the Glu-

284

AgNPs, the –NH2 & -NH stretching vibration of ANDPA decreased and new peaks were

285

appeared at 3394 & 1681 cm-1 which correspond to -OH and –C=O stretching frequencies of

286

glucose molecule attached with AgNPs. The intensity of -C=C stretching frequencies was also

287

slightly diminished and the new peaks were observed at 2384, 1444, 1537 &1566 cm-1. These

288

changes were also observed for =C-H stretching and new peaks were appeared at 2294, 2970 &

289

3047 cm-1. Further, the stretching frequency at 1620, 1681 & 1384 cm-1 indicates the presence of

290

Glu-AgNps. All the results clearly convince the interaction of ANDPA with the surface of Glu-

291

AgNPs (Figure 4c).

292

(Figure 4.)


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In the UV-Visible absorption spectra, the characteristic SPR peak of Glu-AgNPs at 409 nm

294

gradually decreased with obvious red shift to 418 nm due to the Internal Charge Transfer Process

295

(ICT) (Figure 5). The quenching mechanism is mainly due to the donation of electrons from the

296

surface of the Glu-AgNPs to the excited state of ANDPA. The pyridimine nitrogens has high

297

binding effiency with Glu-AgNPs surface due to intermediate base - soft acid (AgNPs)

298

interaction based on Pearson’s hard and soft acids and bases theory. Optimization studies like

299

effect of nature and volume of reducing agent and reduction time were tested for the both Glu-

300

AgNPs and Glu-AgNPs/ANDPA and the details are given in the supplementary (Figure S4-S6).

301

(Figure 5.)

302 303

Glu-AgNPs/ ANDPA conjugate based immunosensor for Pseudomonas aeruginosa

304

Emission studies of Glu-AgNPs/ ANDPA conjugate:

305

As mentioned above, ANDPA exhibited three intense emission peaks at 402, 424 and 446 nm

306

and when ANDPA was attached with the surface of Glu - AgNPs, the emission intensity of the

307

above peaks decreased (Figure 5a). On increasing the concentration of the Glu - AgNPs from

308

0.2 to 6% v/v, the emission intensity of ANDPA was almost diminished. The phenomenon is due

309

to transfer of electrons from the surface of the Glu - AgNPs to excited ANDPA molecule. The

310

optimum time for the binding of ANDPA with the Glu - AgNPs is 20 mins. All other spectral

311

date corresponds to incubation time of ANDPA on Glu-AgNPs surface and optimization of

312

volume of Glu-AgNPs for incubation was given in supporting material (Figure S7 & S8).

313

Attachment of the IgG antibody on Glu-AgNPs/ANDPA:

314

The initial fluorescent intensities of the peaks at 409, 424 and 446 nm of ANDPA in Glu-

315

AgNPs/ANDPA conjugate were fixed for PA recognition study. After the incubation of Ab with

316

Glu-AgNPs/ANDPA conjugate, a gradual increment in fluorescent intensities were observed at

317

409, 424 and 446 nm of ANDPA and almost reached initial fluorescent intensities at the end of

318

the interaction of Ab with Glu-AgNPs/ANDPA (Figure 5b.). Since, the Ab was binding with the

319

Glu-AgNPs/ANDPA, the aggregation process was initiated and thereby all the Glu-

320

AgNPs/ANDPA conjugates come little closure to each other. Covering the whole Glu-AgNPs

321

surface by the Abs could resist the electron donating nature of the nano surface to the excited

322

state of the fluorophore. Therefore, once again the emission intensity increased. This 11 ACS Paragon Plus Environment


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323

phenomenon is further supported by the SEM and TEM images (Figure 7). The synthesized Glu

324

- AgNPs / ANDPA has showed a very good affinity towards like Ab of Pseudomonas

325

aeruginosa. The effect of concentration and volume of bulk Ab and incubation time on

326

fluorescence response and also specific recognition of Ab were shown in the supporting

327

information (Figure S8-S11).

328

Recognition of Pseudomonas aeruginosa (Ab/Glu-AgNPs/ANDPA) conjugates:

329

Generally, the monocolonal Ab have tendency to form a complex with the target PA specifically.

330

Different concentrations of PA were added from 8 to 10-1 CFU/mL with Ab/Glu-

331

AgNPs/ANDPA conjugates and incubated. An enormous increment in fluorescent intensity of

332

peaks at 409, 424 and 446 nm was observed (Figure 6a & 6b). The third peak at 446 nm was

333

red shifted (18 nm) to 468 nm. When the size of the aggregates was increased, a broad red

334

shifted emission band was already observed with increased in intensity. The broad emission is

335

originated from the excited intramolecular charge transfer (ICT) state.49 So, the spectral data are

336

clearly revealing that this is mainly due to the aggregated induced emission of the Glu-AgNPs-

337

ANDPA by the formation of Ab-PA complex. So, all the Ab/Glu-AgNPs/ANDPA conjugates

338

come into very closure distance. This interaction leads to the formation of aggregates or dimer.50

339

The limit of detection was found to be 1.5 CFU/mL and details are given in supporting

340

information (Figure S12). (Figure 6.)

341 342

Interference

of

human

pathogens

with

Pseudomonas

aeruginosa

on

Ab/Glu-

343

AgNPs/ANDPA:

344

In order to confirm the selectivity and specificity of the developed immunoassay, the emission

345

responses of ANDPA in Ab/Glu-AgNPs/ANDPA towards other interference of human pathogens

346

like Salmonella paratyphi A, Klebsiella pneumoniae, Staphylococcus Aureus, Escherichia Coli

347

Pseudomonas Stutteri were checked. The results clearly support that the developed

348

immunosensor has an excellent selectivity and specificity only towards Pseudomonas aeruginosa

349

in presence of above interfering pathogens (Figure 6c). In case of the other interfering

350

pathogens, the emission intensity of ANDPA in Ab/Glu-AgNPs/ANDPA was slightly quenched

351

instead of enormous increase as in the presence of Pseudomonas aeruginosa and this might be

352

due to very weak non specific interaction of the interfering pathogens with the glucose molecules 12 ACS Paragon Plus Environment

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on the silver nanoparticle surface. As a result, the fluorescent molecules get scattered so that the

354

aggregation or dimerization process is arrested and hence quenching in emission intensity is

355

observed.

356

Characterization of Glu-AgNPs, (Ab/Glu-AgNPs/ANDPA) and (PA/Ab/Glu-AgNPs/

357

ANDPA ) SEM and TEM:

358

The structural changes of the Glu-AgNPs, Ab/Glu-AgNPs/ANDPA and PA/Ab/Glu-

359

AgNPs/ANDPA were characterized using SEM and TEM images. From the figure 7a & 7d, it is

360

clearly reveals that the synthesized Glu-AgNPs are cubical in shape with an average particle size

361

of 10 nm. When the antibody (Ab) is immobilized on the Glu-AgNPs/ANDPA, the size of Glu-

362

AgNPs/ANDPA is increased average 250 nm due to slight aggregation (Figure 7b & 17e). After

363

the formation of antibody (Ab):pathogen(PA) complex, the size of Glu-AgNPs/ANDPA is

364

further increased to average 500 nm due to the grater aggregation among PA/Ab/Glu-

365

AgNPs/ANDPA conjugates (Figure. 7c & 7f). (Figure 7.)

366 367 368

Color changes observation under Transilluminator

369

Initially, ANDPA exhibits bluish green fluorescent color. After tagging with the Glu-AgNPs, the

370

fluorescence color was diminished. The color of the probe was totally changed to green

371

fluorescence while incubating the Glu-AgNPs/ ANDPA with Ab and then with PA .This color

372

changes are further supporting the aggregated induce emission due to the Ab:PA complex

373

formation (Figure. 8).

374

(Figure 8).

375

Life time measurement of the immunoassay

376

In order to support the mechanism, the life time measurement was carried out for proposed

377

immunoassay. Initially, the probe showed three emission peaks at 409, 424 & 454 nm. In our

378

current report, the time-correlated single photon counting (TCSPC) analysis of the fluorescence

379

lifetime was utilized in order to support the mechanism. The Time-resolved fluorescence decay

380

was monitored for ANDPA at 409, 424 & 454 nm individually with excitation from nano LED in

381

phosphate buffer at pH =7.0 at 25 °C. The fluorescence decays are fitted to biexponential curve. 13 ACS Paragon Plus Environment


Page 14 of 36

382

The lifetime study details are given in Supporting Information(s). ANDPA has exhibited a

383

lifetime of τ1 = 602 ps and τ2 = 6.19 ns and τ3 = 14 ns with B1 = 0.467, B2 = 0.018 and B3 =

384

0.003 at 409 nm, τ1 =642 ps and τ2 = 4.5 ns and τ3 = 11 ns with B1 = 0.196, B2 = 0.028 and B3

385

= 0.011 at 424 nm and τ1 = 780 ps and τ2 = 4.8 ns and τ3 = 12 ns with B1 = 0.161 B2 = 0.0.027

386

and B3 = 0.008 at 454 nm due to the presence of antharacene moiety. Upon addition of Glu-

387

AgNPs, the lifetime of the initial lifetime for ANDPA has decreased to a minimum value of τ1 =

388

565 ps and τ2 = 4.8 ns and τ3 = 10 ns with B1 = 0.347, B2 = 0.022 and B3 = 0.010 at 409 nm, τ1

389

= 597 ps and τ2 = 4.6 ns and τ3 = 10 ns with B1 = 0.507, B2 = 0.021 and B3 = 0.011 at 424 nm

390

and τ1 = 640 ps and τ2 = 4.6 ns and τ3 = 11 ns with B1 = 0.444, B2 = 0.023 and B3 = 0.010 at

391

454 nm. Finally, incubating the Ab and PA with the lifetime of the Glu-AgNPs/ANDPA was

392

increased to a maximum value of τ1 = 702 ps and τ2 = 4.88 ns and τ3 = 11.7 ns with B1 = 0.234,

393

B2 = 0.029 and B3 = 0.010 at 409 nm, τ1 = 669 ps and τ2 = 4.7 ns and τ3 = 11.4 ns with B1 =

394

0.335, B2 = 0029 and B3 = 0.011 at 424 nm and τ1 = 862 ps and τ2 = 4.15 ns and τ3 = 10.9 ns

395

with B1 = 0.146, B2 = 0.033 and B3 = 0.014 at 454 nm. (Figure S13) All these lifetime

396

measurements were clearly supports the TURN OFF – ON mechanism based on the aggregated

397

induced phenomenon of the immunosensor. [Details are given in supporting information]

398 399

Real sample analysis of P. aeruginosa in water and agricultural products

400

To test the feasibility of the sensor in water, the proposed immunosensor was applied for

401

detection of Pseudomonas aeruginosa in water, soil and three different food items: cow milk,

402

sugarcane and orange juices.50 The same aggregation induce emission responses were

403

obtained for the all real samples when PA inoculated real samples were added with

404

Ab/Glu-AgNPs/ANDPA (Figure 9). The recovery rate was also calculated from fluorimetric

405

responses for all the real samples except soil sample and results are given in the tables 1-

406

4. Since, the soil is the one of the main sources; PA was detected directly from soil samples

407

and their results are given in table 5. The results obtained in the fluorimetric responses were

408

compared with the conventional plate counting method and the results were found to be in

409

good agreement.

410

(Figure 9.) Table 1, 2, 3, 4 & 5

411

and it’s clearly sounds that the proposed immunosensor could be successfully designed and

412

employed for Pseudomonas aeruginosa in all above real samples. 14 ACS Paragon Plus Environment

Page 15 of 36


413

We have adopted a green and facile protocol for the synthesis of pyrimidine fluorophore and

414

AgNPs. Then, a very simple immunosensor platform has been developed from the pyrimidine

415

fluorophore tagged Glucose stabilized AgNPs as label for monoclonal antibody IgG. This

416

developed immunoassay has showed an excellent selectivity towards PA under optimized

417

conditions. The immunosensor could detect the PA from 8 to 10-1 CFU/mL and the Limit of

418

detection was found to be 1.5CFU/mL. The real potent application of the immunosensor was

419

tested for water samples, agricultural soil and food matrices like milk, sugarcane and orange

420

juices succefully.

421

■ Abbreviations

422

ANDPA - (R)-4-(anthracen-9-yl) -6- (naphthalen-1-yl)-1,6-dihydropyrimidine-2-amine

423

Glu-AgNPs – Glucose stabilized silver nanoparticles

424

PA

- Pseudomonas aeruginosa pathogen

425

Ab

- Antibody

426

IgG

- Immunoglobulin G

427

TLC

- Thin Layer Chromatography

428

CFU

- Colony Forming Units

429

TCSPC

- Time-Correlated Single Photon Counting

430

LB

- Luria Bertani

431

TLTC

- To Low To Count

432

TNTC

- To Numerous To Count

433 434

■Acknowledgement:

435

This work is financially supported by DST-INSPIRE Fellowship Scheme, New Delhi, India. So,

436

the author thanks DST for offering the fellowship for pursuing his Ph.D program. The author

437

specially thanks DST-SERB for additional financial support for the same. The author sincerely 15 ACS Paragon Plus Environment


Page 16 of 36

438

thanks DST IRHPA, DST-PURSE and School of Chemistry, Madurai Kamaraj University for

439

providing the NMR, SEM, TEM and other common instrument facilities.

440

■ASSOCIATED CONTENT

441 442

Supporting Content

443 444

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

445

Characterization of ANDPA: NMR & ESI-Mass Spectra, UV and Fluorescence Optimization

446

studies for the developed immunoassay.

447 448

■Author Information

449

* Corresponding Author

450

[email protected]

451

Fax: + 91 - 452 - 245 8449

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468


Page 17 of 36

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653 654

■ Figure Captions

655

Figure 1. (a). Synthetic route for ANDPA fluorophore and (b). Glucose stabilized Ag NPs.

656

Figure 2. (a). UV visible characterization spectra and (b). Corresponding emission spectra for

657

ANDPA. [Concentration of ANDPA is 5 µM (pH=7.0 PBS Buffer)]

658

Figure 3. (a) Emission spectral changes while varying the pH from 2 to 13. (b) Corresponding

659

linear plot. [Concentration of ANDPA is 5 µM, ANDPA was prepared from the stock solution

660

of 1 mM of ANDPA under different pHs using various buffers.]

661

Figure 4. (a) SPR band of synthesized Glu-AgNPs and (b) XRD analysis of synthesized Glu-

662

AgNPs. (c) IR spectra for the synthesized ANDPA and Glu-AgNPs/ANDPA. [Concentration of

663

Glu-AgNPs: 6 %(v/v)]

664

Figure 5. (a) Emission property of ANDPA with and without Glu - AgNPs under optimized

665

condition. (b) Emission studies for the binding of antibody (Ab) with Glu-AgNPs/ANDPA under

666

optimized condition. [Concentration of ANDPA is 5 µM, Glu-AgNPs: 6 %(v/v), Ab: 0.36

667

µg/mL(20 µL) in pH=7.0 (PBS Buffer)]

668

Figure 6. (a) Emission studies for the binding of PA with Ab/Glu-AgNPs/ANDPA under

669

optimized condition. (b) Colony variation of PA from 8 to 10-2 CFU/mL (c) Emission studies

670

for the selective binding of PA with Ab/Glu-AgNPs/ANDPA under optimized condition in

671

presence of other interfering pathogens. [Concentration of ANDPA is 5 µM, Glu-AgNPs: 6

672

%(v/v), Ab: 0.36µg/mL(20 µL) and PA and other pathogens are 10-7 µg/mL(40 µL) in pH=7.0

673

(PBS Buffer)]

674

Figure 7. SEM(a-c) and TEM(d-f)

675

AgNPs/ANDPA (b & e) and PA/Ab/Glu-AgNPs/ANDPA [Concentration of ANDPA is 5 µM,

676

Glu-AgNPs: 6 %(v/v), Ab: 0.36 µg/mL(20 µL) PA: 10-7 µg/mL(40 µL) in pH=7.0 PBS Buffer)].

analysis for the Glu-AgNPs/ANDPA (a &d), Ab/Glu-



Page 24 of 36

677

Figure 8. Transilluminator images: Synthesized fluorophore ANDPA (a), Glu-AgNPs/ANDPA

678

(b) and PA/Ab/ Glu-AgNPs/ANDPA(c).

679

Figure 9. Real time analysis of Pseudomonas aeruginosa based on the fluorescence responses of

680

Ab/ Glu-AgNPs/ANDPA in water (a), milk (b) sugarcane juice (c), orange juice (d) and soil (e)

681

[Concentration of ANDPA is 5 µM, Glu-AgNPs: 6 %(v/v), Ab: 0.36 µg/mL(20µL), spiked real

682

samples (40 µL): containing 8, 31, 60, 122 & 265 CFU/mL of PA and soil samples (40 µL) under

683

different dilutions factor from 10-5 – 10-9].

684

Table 1. Comparison of results obtained for the detection Pseudomonas aeruginosa in water

685

samples using the plate count method and current developed fluorescence immunoassay

686

protocol. [Concentration of ANDPA is 5 µM, Glu-AgNPs: 6 %(v/v), Ab: 0.36 µg/mL(20 µL) ,

687

Spiked water samples (40 µL); containing 8, 31 & 60 CFU/mL of PA].

688

Table 2. Comparison of results obtained for the detection Pseudomonas aeruginosa in milk

689

using the plate count method and current developed fluorescence immunoassay protocol.

690

[Concentration of ANDPA is 5 µM, Glu-AgNPs: 6 %(v/v), Ab: 0.36µg/mL(20µL) , Spiked milk

691

samples (40 µL) containing 31, 60, 122 & 265 CFU/mL of PA].

692

Table 3. Comparison of results obtained for the detection Pseudomonas aeruginosa in sugar

693

cane juice using the plate count method and current developed fluorescence immunoassay

694

protocol. [Concentration of ANDPA is 5µM, Glu-AgNPs: 6 %( v/v), Ab: 0.36µg/mL (20 µL),

695

Spiked sugarcane juice samples (40 µL) containing 8, 31, 122 & 265 CFU/mL of PA].

696

Table 4. Comparison of results obtained for the detection Pseudomonas aeruginosa in

697

orange juice using the plate count method and current developed fluorescence

698

immunoassay protocol. [Concentration of ANDPA is 5 µM, Glu-AgNPs: 6 %( v/v), Ab: 0.36

699

µg/mL (20 µL), Spiked orange juice samples (40 µL) containing 8, 31, 122 & 265 CFU/mL of

700

PA].

701

Table 5. Detection of Pseudomonas aeruginosa in soil sample using current developed

702

fluorescence immunoassay protocol and comparison with the plate count method.

703

[Concentration of ANDPA is 5 µM, Glu-AgNPs: 6 % (v/v), Ab: 0.36µg/mL (20µL), soil samples

704

(40 µL) under different dilutions factor from 10-5 – 10-9]. 24 ACS Paragon Plus Environment

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705 706

Tables Sl.no

Water Source

Added (CFU/mL)

Found (CFU/mL)

Recovery (%)

Plate count method (CFU/mL)

1

Madurai

8

8

100

9

2

Dindigul

31

29

94

31

3

Salem

8

7

88

8

4

Virudhunagar

60

57

95

60

5.

Chennai

31

18

58

20

Recovery (%)


92 102 108 106

252 110 51 35

707 708

Table 1.

709

Sl.no

Added (CFU/mL)

1 2 3 4

265 122 60 31

Found (CFU/mL) 245 125 65 33 Table 2.

710 711

712

Sl.no

Added (CFU/mL)

Found (CFU/mL)

Recovery (%)


1 2 3 4

265 122 31 8

240 115 30 7

94 94 96 87

245 112 25 5 (TLTC)

Table 3.

713



Sl.no

714

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Added (CFU/mL)

Found (CFU/mL)

Recovery (%)


1 2 3 4 Table 4.

265 122 31 8

230 140 25 8

86 114 80 100

245 115 37 7(TLTC)

Sl.No.

Agricultural Soil

This method (unknown) (CFU/mL)

Plate count Method (unknown) (CFU/mL)

1. 2. 3. 4. 5.

Test 1 Test 2 Test 3 Test 4 Test 5

275 135 50 26 10

320(TNTC) 180 70 45 17

715

716 717 718 719 720 721 722 723 724 725


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726


■ Figure Graphics 727 728

729

Figure 1.

730 731 732 733 734 735 736 737 738 739 740 741 742 743 744



745

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Figure 2.

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762

763

Figure 3.

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Figure 4.

797 798 799 800 801 802 803


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804 805 806 807 808 809 810

811

Figure 5.

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Figure 6.

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842 843 844 845 846 847 848 849 850 851 852 853 854 855 856

Figure 7.

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Figure 8.

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Figure 9. 35 ACS Paragon Plus Environment


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■ Graphic for table of contents

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For TOC only

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“Simple Immunoassay for selective recognition of Pseudomonas Aeruginosa”

915 916 917 918 919 920 921 922


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