AuNPs-LISA, an efficient detection assay for Opisthorchis viverrini (Ov) antigen in urine

Journal Pre-proof AuNPs-LISA, an efficient detection assay for Opisthorchis viverrini (Ov) antigen in urine Wichit Taron, Wassana Jamnongkan, Anchalee Techasen, Jutarop Phetcharaburanin, Nisana Namwat, Paiboon Sithithaworn, Narong Khuntikeo, Siriboon Mukdasai, Somphou Sayasone, Watcharin Loilome, Wittaya Ngeontae PII:

S0039-9140(19)31225-1

DOI:

https://doi.org/10.1016/j.talanta.2019.120592

Reference:

TAL 120592

To appear in:

Talanta

Received Date: 13 September 2019 Revised Date:

21 November 2019

Accepted Date: 24 November 2019

Please cite this article as: W. Taron, W. Jamnongkan, A. Techasen, J. Phetcharaburanin, N. Namwat, P. Sithithaworn, N. Khuntikeo, S. Mukdasai, S. Sayasone, W. Loilome, W. Ngeontae, AuNPs-LISA, an efficient detection assay for Opisthorchis viverrini (Ov) antigen in urine, Talanta (2019), doi: https:// doi.org/10.1016/j.talanta.2019.120592. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Graphical Abstract

1

AuNPs-LISA, an efficient detection assay for Opisthorchis

2

viverrini (Ov) antigen in urine

3

Wichit Taron a , Wassana Jamnongkan b, Anchalee Techasen b,c, Jutarop

4

Phetcharaburanin a,b, Nisana Namwat a,b, Paiboon Sithithaworn b,d, Narong Khuntikeo

5

b,e

6

Ngeontae f**

7 8

a

9

b

Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand

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c

Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

11 12

d

13

e

14 15

f

16 17 18

g

, Siriboon Mukdasai f, Somphou Sayasoneg, Watcharin Loilome a,b*, Wittaya

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand

Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand Lao Tropical and Public Health Institute, Vientiane Capital, Lao People's Democratic Republic

19 20 21

Corresponding authors:

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W. Ngeontae**

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Tel.: +66-430-09700x42174, Fax.: +66-432-02373

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Email address: [email protected]

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W. Loilome*

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Tel.:+66-433-63265, Fax.: +66-433-63265

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Email address: [email protected]

28

Abstract

29

The enzyme-linked immunosorbent assay (ELISA) is currently a powerful technique for

30

the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, its sensitivity

31

and analysis time need to be improved. In the present study, we aimed to improve the signal

32

enhancing system of traditional ELISA by using gold nanoparticles (AuNPs) with peroxidase-like

33

activity on its surface instead of the horseradish peroxidase (HRP) system. The catalytic activity of

34

the AuNPs probe can be boosted by the gold enhancing solution and the addition of ATP. The

35

catalytic ability of the AuNPs probe depended on the probe and the H2O2 concentration. The

36

proposed approach can reduce the number of the traditional ELISA steps with better detection

37

sensitivity. Interestingly, the limit of detection (LOD) of the test was 23.4 ng mL-1, substantially

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lower than the 93.8 ng mL-1 for the traditional ELISA. The AuNPs-LISA assay showed higher

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sensitivity and specificity, 93.81% and 91.34%, respectively, compared to the traditional ELISA.

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The proposed assay was successfully applied for the detection of OvAg in urine samples. This will

41

provide an effective tool for the detection, control and elimination of human opisthorchiasis.

42

Keywords: Opisthorchiasis, Gold nanoparticles (AuNPs), Cholangiocarcinoma (CCA), Peroxidase-like

43

activity, Nano ELISA

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1. Introduction

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The Greater Mekong Subregion (GMS) of Southeast Asia, particularly the northeast

46

of Thailand and the Lao People’s Democratic Republic (Lao PDR), has long been known to

47

have the highest incidence of cholangiocarcinoma (CCA) worldwide [1]. The most important

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risk factor associated with CCA in this region is liver fluke infection caused by Opisthorchis

49

viverrini (Ov), which is acquired through eating raw or inadequately cooked (salted, pickled,

50

or smoked) infected cyprinid fish [2]. In the GMS region, an estimated 40 million people

51

(20% of the population) are currently infected with Ov [1,3,4]. Ov infection is most common

52

in the adult population, particularly males [5,6]. In addition, more than 600 million people are

53

at risk of liver fluke infection [7], indicating that simple, cheap and reliable diagnostic

54

methods are urgently required.

55

The formalin-ethyl acetate concentration technique (FECT) is the most common

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diagnostic technique and is defined as a gold standard for the determination of Ov eggs in

57

fecal samples [8,9]. However, this technique has several disadvantages including low

58

sensitivity, being time-consuming and requiring a qualified microscopist to differentiate the

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Ov eggs from those of other helminth parasites, which are similar in shape and size [10–12].

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In addition, in cases of light infections may be difficult to detect and biliary tract obstruction

61

acts as a barrier of the flow of Ov eggs into feces, which it is a drawback for Ov eggs

62

detection by FECT; false-negatives result maybe occur [6,13].

63

Recently, Ov antigen detection in urine samples was developed using the enzyme-

64

linked immunosorbent assay (ELISA) [14]. Worasith and co-workers used monoclonal

65

antibody (IgG1 murine mAb, clone KKU505) to coat ELISA plates. This combined with

66

OvAg and using a protein A purified rabbit IgG against OvAg formed a sandwich structure

67

[14–16]. The signal enhancement of this model used the HRP system, which is one of the

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most common enzymes used for ELISA [17]. This model has been reported to have higher

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diagnostic sensitivity and specificity (81% and 71%, respectively) than FECT for detecting

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Ov infection [14]. Moreover, it is easy to use and the urine is collected noninvasively easing

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sample collection. However, this model still has some shortcomings, such as its detection

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sensitivity, its multi-step procedure, it is also time-consuming taking at least 6 hours, which

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does not include the coating step. In addition HRP enzyme is easily denatured and digested

74

by proteases and its preparation is complicated [14,18].

75

A potentially interesting new approach involves enzyme-like catalytic activities such

76

as peroxidase and catalase that have been demonstrated on the surface of nanomaterials such

77

as gold nanoparticles (AuNPs) [19,20,21,22,23], CuO nanoparticles [13], Co3O4

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nanoparticles [24], and Fe3O4 nanoparticles [25]. In particular, AuNPs have been reported

79

with peroxidase-like activity, which could catalyze the oxidation of H2O2 to produce

80

hydroxyl radicals. Further reaction of the hydroxyl radicals could change the chromogenic

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substrate from colorless, e.g. using OPD and TMB, to a colored product of benefit to

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bioanalysis and diagnostics in the field of medical science [26–30].

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Unfortunately, if the surface of AuNPs is conjugated with proteins, its peroxidase-like

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activity is decreased or almost inhibited [30,31]. However, the catalytic activity can be

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recovered by several strategies such as the formation of an amalgam with Hg2, which

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stimulates the peroxidase-like activity of AuNPs graphene oxide (AuNPs-GO) hybrids for

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respiratory syncytial virus (RSV) detection based on colorimetric immunoassay [31].

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Moreover, the gold enhancement solution has been used to initiate the catalytic activity of

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AuNPs with conjugated proteins because the surface of AuNPs is coated with gold growth

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solution, similar to the seed-mediated nucleation of AuNPs synthesis [30,32,33]. Previous

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reports suggest that biomolecules such as ATP, ADP can enhance the peroxidase-like activity

92

of AuNPs via the stabilization of oxidized TMB [19,28]. The peroxidase-like activity of

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AuNPs is dependent on pH, temperature, H2O2 concentration, size, shape and surface charge

94

[26,28,34,35]. For example, increasing the H2O2 concentration strongly enhanced the

95

intrinsic peroxidase-like activity of AuNPs [35]. AuNPs have been utilized in the signal

96

amplification of ELISA-like assays. Their catalytic activity showed a good linear relationship

97

in the range of 0.7-100 ng mL-1 and LOD of 0.3 ng mL-1 in the IgM analytical model, which

98

is better than HRP using the conventional sandwich ELISA [14]. The peroxidase-like activity

99

of AuNPs may be utilized to solve some of the shortcomings of the traditional ELISA for Ov

100

detection.

101

In this work, we proposed a new strategy to detect the OvAg in urine samples by

102

improving the drawbacks of the traditional ELISA. The enzyme HRP, which always used in

103

the color developing step of the traditional ELISA, was replaced by the peroxidase-like

104

activity of the AuNPs probe. The improvement of the peroxidase-like activity of the AuNPs

105

probe was demonstrated by using gold enhancing solution and the addition of ATP. The

106

parameters that may affect to the detection sensitivity of the proposed assay were studied and

107

optimized. In order to demonstrate the applicability of the proposed assay, real urine samples

108

from the participants in an endemic area for liver fluke infection in the Pak Ngum district of

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Lao PDR were analyzed and compared with the traditional ELISA. The proposed assay will

110

provide an effective tool for the diagnosis control and elimination of human opisthorchiasis.

111

2. Materials and methods

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2.1. Materials

113

Crude O. viverrini adult excretory secretory antigen (OvAg) and a protein A purified

114

rabbit IgG against crude OvAg (IgG pAb) were supplied by Prof. Paiboon Sithithaworn.

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Streptavidin horseradish peroxidase (HRP) conjugate was purchased from GE Healthcare

116

(UK). Orthophenylenediamine hydrochloride (OPD), adenosine triphosphate disodium salt

117

(ATP), chloroauric acid (HAuCl4•3H2O) and hydroxylammonium chloride (NH2OH•HCl)

118

were purchased from Sigma (USA). Sulfuric acid (H2SO4), sodium hydrogen carbonate

119

(NaHCO3), sodium chloride (NaCl), disodium hydrogen phosphate (Na2HPO4), sodium

120

hydroxide (NaOH) and citric acid monohydrate (C6H8O7•H2O) were purchased from RCI

121

Labscan Ltd.(USA). Goat F(ab’)2 anti-rabbit IgG HL (10 nm Gold) was purchased from

122

Abcam (UK). 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) solution (1X), biotin conjugate,

123

sodium carbonate anhydrous (Na2CO3) and tween-20 were purchased from Thermo Fisher

124

Scientific (USA). Hydrogen peroxide (30% H2O2) was purchased from Merck (Germany).

125

Sodium dihydrogen phosphate dihydrate (NaH2PO4•2H2O) and sodium dihydrogen

126

phosphate monohydrate (NaH2PO4•H2O) were purchased from QRec (New Zealand). SKM

127

growth powder was purchased from HiMedia Laboratories Pvt. Ltd. (India). All reagents

128

were of analytical grade.

129

The buffers used in this study were: 50 mM bicarbonate buffer pH 9.6, containing

130

Na2CO3 and NaHCO3 used as coating buffer; incubating buffer, 0.05% Tween 20 in

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phosphate buffered saline (PBST), pH 7.4 containing NaCl, Na2HPO4, NaH2PO4•2H2O and

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Tween 20; blocking buffer, 5% (w/v) SKM in PBST pH 7.4; washing buffer mixing 1%

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Tween 20 with PBS buffer pH 7.4 and citrate phosphate buffer pH 5.0 containing

134

C6H8O7•H2O and NaH2PO4•2H2O.

135

2.2. Monoclonal antibody

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The monoclonal antibody (IgM mAb, clone KKU505) was previously established

137

[14]. The cloned KKU505 myeloma cell line was cultured in Roswell Park Memorial

138

Institute (RPMI) in 1640 medium with L-glutamine (Corning, USA) supplemented with 100

139

U mL-1 penicillin and 0.1 mg mL-1 streptomycin at 37°C in 5% CO2. The culture medium was

140

collected every 24 h. The secreted mAb in the medium was concentrated by Macrosep

141

Advance centifugal filters 3K (Pall, USA) at 5,000 x g and 4°C for 1 h. Monoclonal antibody

142

was kept frozen at -20 °C until used.

143 144 145 146

2.3. Optimization of ELISA conditions using the AuNPs probe as the signal amplifier 2.3.1. The effect of the enhancing solution on AuNPs probe activity in the ELISA assay

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Goat F(ab’)2 anti-rabbit IgG HL (10 nm Gold) was used as the AuNPs probe’s

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signal amplifier in the ELISA system instead of the HRP system [14]. Monoclonal antibody

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(mAb, IgM, clone KKU505) was diluted with 50 mM bicarbonate buffer, pH 9.6 to 10 µg

150

mL-1 mAb and 100 µL was added to flat-bottom 96-well microtiter plates (NUNC, Roskilde,

151

Denmark). The sealed plates were incubated at 4 oC overnight. Then, the coated plates were

152

washed with washing buffer 3 times on the shaker at 700 rpm for 30 s/time. Next, 200 µL of

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blocking buffer were added to each well and kept for 1 hr at 37oC after which the plates were

154

washed for 3 times. Next, 100 µL of PBS diluted crude OvAg (12 µg mL-1) was added to the

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wells in duplicates, and incubated at 37 oC for 2 hrs. After washing 5 times, 100 µL of 7 µg

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mL-1 IgG pAb in 2%SKM incubating buffer was added to the wells which were kept for 1 hr

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at 37 oC. After washing 3 times, 10 systems using the signal enhancing step were optimized:

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100 µL of goat F(ab’)2 anti-rabbit IgG HL (10nm gold) (Abcam, UK) as AuNPs probe were

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added to each well for all systems, except 9 and 10, with 100 µL/well of deionized water (DI)

160

and incubated at 37oC for 1 hr. The plates were washed 3 times and the color development

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observed: System 1: neither chemical was added, but TMB-H2O2 solution was pipetted into

162

wells, system 2: only 1X TMB solution was added, system 3: only H2O2 was added, system

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4: both chemicals mixed with TMB-H2O2 were added. In systems 5-8 100 µL Au enhancing

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solution 1:1 (v:v) mixture of 5 mM HAuCl4•3H2O : 10 mM NH2OH•HCl) was added to the

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wells and incubated for 20 min at room temperature using the method of Kim et al. [14,32].

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This was followed by washing 5 times with distilled water. The color developing step was not

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added to system 5 whereas the TMB chromogenic substrate only was added to system 6.

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Only H2O2 was added to the system 7. The both chemicals were added to the wells of system

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8. Au enhancing solution was added to system 9 but not to system 10. Both of these systems

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included the TMB chromogenic substrate. After adding TMB chromogenic substrate, the

171

plates were incubated at 37oC in the dark for 15 min. The blue color appeared and changed to

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yellow by adding 50 µL of 4N sulfuric acid. This was measured by using an Agilent HP 8453

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UV-vis spectrophotometer (Waldbronn, Germany), which scanned wavelengths from 300 and

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800 nm.

175 176

2.3.2. AuNPs probe and H2O2 concentration effects the signal enhancement of AuNPs-LISA

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The AuNPs probe was diluted with DI water to 1:480, 1:240, 1:120, 1:60 and

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1:30 dilutions. In brief, after IgG pAb was added, 100 µL of each AuNPs probe concentration

179

was added to wells and incubated at 37 oC for 1 hr. After washing 3 times, 100 µL of the Au

180

enhancing solution (1:1, 5 mM HAuCl4•3H2O : 10 mM NH2OH•HCl, v:v) was added to the

181

wells and incubated at room temperature for 20 min. After washing 5 times with DW, 150 µL

182

of the mixed TMB-H2O2 solution was added to the plates and incubated at 37 oC for 15 min.

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The blue color changed to yellow by adding 50 µL of 4N sulfuric acid, and the optical

184

density (OD) was measured at 450 nm using a Tecan sunrise microplate reader (Tecan,

185

Crailsheim, Germany).

186

For the next step, H2O2 concentrations were optimized (0.8, 1.5, 3, 6 and 12%

187

H2O2) in the mixed TMB chromogenic substrate. After adding the enhancing solution and

188

washing 5 times, the mixed TMB chromogenic substrate with different H2O2 concentrations

189

was added to wells and kept for 15 min at 37oC. Then, 50 µL of 4N sulfuric acid was added

190

and the OD measured as described above.

191

2.3.3. The effect of ATP on signal enhancement in the AuNPs-LISA assay

192

ATP solution was prepared using the Shah and Singh method [28]. Briefly, 5

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mM ATP solution was freshly prepared before using 0.1275 g ATP dissolved in 50 ml DI

194

water. ATP solution was used to increase the colorimetric TMB signal of the AuNPs-LISA.

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This step was optimized. For the 4 systems containing a colorimetric TMB step, 150 µL of

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mixed 1X TMB containing H2O2 with the volume adjusted using DI water was added to

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system 1, 50 µL of 5 mM ATP and 150 µL of mixed TMB-H2O2 solution were added to

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system 3. After the Au enhancing solution step, 150 µL of mixed TMB-H2O2 solution was

199

added to wells of system 2 and the volume adjusted with DI water, while for system 4 50 µL

200

of 5 mM ATP and 150 µL of mixed TMB-H2O2 solution were added. After incubation for 15

201

min at 37 oC, the blue color appeared and changed to yellow after adding 50 µL of 4N

202

sulfuric acid. The OD was measured at 450 nm using a Tecan sunrise microplate reader

203

(Tecan, Crailsheim, Germany) in the presence and absence of ATP. The ∆OD ([OD at 450

204

nm in the presence of ATP] - [OD at 450 nm in the absence of ATP]) was plotted as a

205

function of the concentration of ATP.

206

For further optimization, the ATP concentration was serially diluted to 0.078,

207

0.156, 0.313, 0.625, 1.25 and 2.5 nm ATP in DI water. Following this, 50 µL of diluted ATP

208

was added to the wells, followed by 150 µL of mixed TMB-H2O2 solution and incubated for

209

15 min at 37 oC. The OD was measured as described above.

210

2.4. The Limit of Detection (LOD)

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OvAg was added to phosphate buffered saline (PBS), pH 7.4, and serially diluted to

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12,000, 6,000, 3,000, 1,500, 750, 375, 187.5, 93.8, 46.9, 23.4, 11.7 and 5.9 ng mL-1. The

213

plates were coated, washed, blocked and washed again as described in Section 2.3.1. The

214

diluted OvAg was added to the wells and incubated for 2 hr at 37 oC. Then the plates were

215

carefully washed 5 times with washing buffer and 100 µL of IgG pAb were carefully added

216

to the wells which were ten kept at 37 oC for 1 hr before being washed 3 times.

217

For the traditional ELISA method [14], 100 µL of 1:2,000 diluted conjugated-biotins

218

in incubating buffer were added to the wells and kept at 37 oC for 1 hr. 100 µL of diluted

219

1:2,500 conjugated-HRP in incubating buffer were added after washing 3 times and kept at

220

37oC for 1 hr. The substrate solution, OPD with 0.03% H2O2, was added to the wells which

221

were kept in the dark at room temperature for 15 min. Following this, 100 µL of 4N sulfuric

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acid were added to the wells to stop the reaction and the OD was measured at 492 nm.

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For the AuNPs-LISA assay, 100 µL of 1:60 diluted AuNPs probe in incubating

224

buffer were added to the AuNPs-LISA assay plate and kept at 37oC for 1 hr. Following this,

225

100 µL of Au enhancing solution were added to wells after washing 3 times and the plate was

226

kept at room temperature for 20 min. This was followed by washing 5 times with DW after

227

which 50 µL of 5mM ATP in DI water and 150 µL of mixed TMB-H2O2 solution were added

228

to the wells and kept at 37oC for 20 min. The OD was measured as described in Section 2.3.3.

229

Thus, the AuNPs-LISA assay for OvAg detection in the urine sample was conducted using

230

the AuNPs probe instead of the HRP system.

231

2.5. Sample collection

232

Three hundred and ninety urine samples were corrected from the participants in an

233

endemic area for liver fluke infection in the Pak Ngum district of Lao PDR. These were all

234

enrolled in the project “Combating Cholangiocarcinoma in Lao PDR” operated under

235

collaboration between the Cholangiocarnima Research Institute (CARI), Khon Kean

236

University, Thailand and Lao Tropical and Public Health Institute, Lao PDR. First morning

237

mid-stream urine samples were collected in plastic containers and kept at 4–8°C in a cool box

238

during transport to the laboratory. This occurred within 24 hours of sample collection to

239

ensure timely processing. Urine samples were centrifuged at 1,500 rpm at 4°C for 15 min and

240

200 µL of clarified supernatant were aliquoted into 96-well microplates, sealed and stored at -

241

20°C until used in the ELISA assay [14].

242 243

2.6. Traditional ELISA method and AuNPs-LISA for OvAg detection in urine samples

244

The plates were coated, washed, blocked and washed again as described in Section

245

2.3.1. The urine samples from Section 2.5 were centrifuged at 1500 rpm for 5 min at 4oC.

246

Following this, the samples were added to the wells, 100 µL/well in duplicate with PBS.

247

Negative Ov urine served as negative controls while positive Ov urine and OvAg were

248

positive controls. After washing 5 times, 100 µl of 7 µg mL-1 IgG pAb in 2%SKM in

249

incubating buffer were added to the wells that were then kept for 1 hr at 37 oC. Following

250

this, the method described in Section 2.4 for the AuNPs-LISA assay was followed. The OD

251

values of the samples were transformed to a ratio between the OD of samples and the OD of

252

the positive control [14]. The diagnostic performance of the AuNPs-LISA assay was

253

evaluated. An Area under the Curve (AUC) of the Receiver Operating Characteristic (ROC)

254

curve was used to determine the probability of correctly detecting Ov infection between the

255

traditional ELISA method and the AuNPs-LISA assay [14,36]. ROC curve analysis and the

256

Yoden index were established to evaluate diagnostic sensitivity, specificity and the cut-off

257

value of the AuNPs-LISA assay. These values from the ROC analysis were used to further

258

characterize the assay’s performance as follows: the Positive Predictive Value (PPV),

259

Negative Predictive Value (NPV), diagnostic accuracy, Positive Likelihood Ratio (PLR),

260

Negative Likelihood Ratio (NLR) and Disease prevalence (DP) were used to compare the

261

performance of AuNPs-LISA assay with the traditional ELISA method.

262

2.7. Ethical statement

263

Ethical approval was obtained from the National Ethics Committee for Health

264

Research, Ministry of Health (MoH) of Lao PDR (reference no. 098/NECHR). Permission

265

for fieldwork was obtained from the Ministry of Health, the Vientiane Capital Health Office

266

and the Pagneum District Health Office. Before enrolment, a meeting was held with the local

267

authorities. The participants were given detailed explanations about the objectives,

268

procedures, potential risks, and benefits of the study. The written information sheet in Lao

269

language was read to all participants and their questions were answered. Informed consent

270

approval was obtained from all study participants prior to the initiation of the sample

271

collection. Study participants positive for intestinal parasitic infections in the final assessment

272

were treated according to national treatment guidelines (reference: MOH. Diagnosis and

273

treatment at the district hospital. A diagnosis and treatment guideline for the district hospital

274

in Lao PDR. Vientiane: Ministry of Health. 2004).

275

2.8. Data analysis and statistical methods

276

All data were analyzed using SPSS 21 (IBM, Chicago, IL, USA) and MedCal (Med-

277

Calc Software, Ostend, Belgium). Descriptive statistics, e,g., frequency and mean were used

278

to summarise the sample distribution. Likelihood ratio test was applied to examine the

279

association of prevalence of Ov in patients. Sensitivity value, specificity value, positive

280

predictive value and negative predictive value were used to describe the diagnostic accuracy

281

of the AuNPs-LISA. Statistical significance was defined with a P-value < 0.05.

282

3. Results and discussion

283

3.1 Gold-enhanced peroxidase-like activity of the AuNPs probe

284

Basically, the HRP system is used in the traditional ELISA method for OvAg

285

detection in urine [14]. However, this system consists of incubation and washing steps, which

286

take approximately 2 hrs [14]. In order to improve the traditional assay, the HRP system was

287

replaced by the goat F(ab’)2 anti-rabbit IgG HL conjugated with 10 nm gold (AuNPs probe)

288

as shown in Fig. 1. Thus, the color development step was shorted because AuNPs can act as

289

the HRP function. However, when AuNPs are modified with proteins, they may exhibit low

290

peroxidase-like activity [30] due to the protein covering the active surface area of the ultra

291

small AuNPs. To solve this problem, Wang et al. suggested that a gold growth solution be

292

used to recover the peroxidase-like activity of AuNPs [30]. An AuNPs prepared gold-

293

enhanced AuNPs probe can catalyze TMB oxidation in the presence of H2O2 based on the

294

peroxidase-like activity of AuNPs [14,19,26,27,29]. In order to prove this concept, the

295

peroxidase like activity of the AuNPs probe was modified with gold enhancing solution

296

(HAuCl4 and NH2OH) and the AuNPs probe without gold enhancing solution for comparison

297

in the color developing assay in the presence of H2O2 and TMB. The results in Fig. 2A show

298

that the yellow color can be formed only in the system with the AuNPs probe modified with

299

gold enhancing solution in the presence of H2O2 and TMB, for which in Fig 2B shows the

300

reproducibility of system 8. On the other hand, the system consisting of the AuNPs probe and

301

other related systems did not exhibit the peroxidase-like activity of AuNPs. The UV-

302

absorbance spectra of system 8 showed high absorbance at A450 nm. Systems 1-4 confirmed

303

that the peroxidase-like activity of the AuNPs probe was almost completely inhibited when

304

the surface of the AuNPs was conjugated with proteins [30,31]. The enhancing solution could

305

be used to recover the peroxidase-like activity of the AuNPs probe which catalyzed H2O2 to

306

convert the chromogenic substrate TMB to oxTMB with a blue color [30,32] that converted

307

to a yellow color after the addition of acid. The catalytic reaction of peroxidase-like activity

308

on the surface of AuNPs could adsorbed H2O2 and break the O-O bond to generate radicals,

309

which further catalyzed the chromogenic substrate TMB [19,26,27,29,30]. The enhancing

310

solution worked similarly to the seed-mediated synthesis of AuNPs because the surface of

311

AuNP was coated to form a new shell resulting in an increase in the AuNPs’ diameter

312

[30,32,33]. The TEM images of the AuNPs after reaction with gold enhancing solution were

313

examined as the results showed in Fig. S1 (ESI). It was clearly seen that the size of the

314

AuNPs probe were significantly increased after reaction with gold enhancing solution.

315

Therefore, the results from this experiment confirm that the AuNPs probe modified with gold

316

enhancing solution can potentially be used instead of the HRP system of traditional ELISA.

317

Insert Fig. 1 here.

318

Insert Fig. 2 here.

319 320

3.2 The parameters affecting the detection sensitivity of the AuNPs-LISA assay and their optimization

321

To investigate the effect of the AuNPs probe, H2O2 and ATP were used in the

322

proposed AuNPs-LISA assay (with the gold enhancing step). Figure 3A shows the OD values

323

for the reaction solution at A450 nm with different concentrations in the AuNP probe. The

324

OD values in the reaction solution increased gradually according to the increasing

325

concentration of the AuNPs probe until a 1:60 dilution was reached. This result indicates that

326

the peroxidase-like activity of the AuNPs probe in the AuNPs-LISA assay depends on the

327

AuNPs probe concentration [28]. This observation is supported by the result of UV-vis

328

absorbance spectra of the reaction solution that increased to a visually deeper yellow color

329

change (Fig. 3B).

330

Insert Fig. 3 here.

331

As shown in Fig. 3C, the OD values for the reaction solution corresponded with

332

increasing H2O2 concentration up to 6% H2O2 (Fig. 3C), as did the UV-vis absorbance

333

spectra (Fig. 3D). This result indicates that increasing the H2O2 concentration to the reaction

334

to more than 6% H2O2 does not lead to increasing peroxidase-like activity [35]. However, the

335

signal was still of low quality. Therefore, we further studied the effects of other molecules on

336

the peroxidase-like activity of the AuNP probe. There have been reports that the peroxidase-

337

like activity of AuNPs can be enhanced in the presence of ATP [19,28]. As shown in Fig. 4A,

338

the UV-vis absorbance spectra of signal enhancement of AuNPs-LISA in the presence of

339

ATP (blue line) showed a higher absorbance than in the absence of ATP (red line). ATP acts

340

as the linkage between AuNPs and chromogenic TMB that promotes the interaction between

341

hydroxyl free radicals and TMB to generate oxTMB, showing enhanced peroxidase activity

342

[19,28]. It has been reported that the peroxidase-like activity of the AuNPs probe could

343

catalyze the oxidation of the TMB chromogenic substrate by H2O2 as an electron acceptor to

344

develop a blue color [30]. It seems that high concentrations of ATP do not influence the

345

stability of oxidized TMB as show in Fig. 4B. The OD value of the reaction solution was

346

decreased when the ATP concentration was increased by more than 1.25 mM in the AuNPs-

347

LISA assay. The results from this experiment strongly confirm that the peroxidase-like

348

activity of the gold enhanced-AuNPs probe can be enhanced by the simple addition of ATP.

349

Thus, in order to obtain the best detection sensitivity, ATP is then added to the detection

350

assay to the concentration of 1.25 mM. Although the concentration of ATP at 0.625 mM

351

provided a comparative sensitivity to 1.25 mM ATP, some of the ATP may partially degrade

352

at room temperature. Therefore, the ATP concentration at 1.25 mM was used in order to

353

obtain the best detection sensitivity. Thus in order to make sure that the ATP is sufficient to

354

provide the highest catalytic activity, the concentration of ATP at 1.25 mM was chosen.

355

356

Insert Fig. 4 here. 3.3 Analytical merits of the AuNPs-LISA assay

357

In order to compare the detection sensitivity of the AuNPs-LISA assay with the

358

traditional ELISA assay, the detection limits (LOD) of both systems are determined by

359

measuring the OD of the solution assay when the concentration of OvAg is decreased as the

360

results show in Fig. 5. The LOD of the AuNPs-LISA assay was 23.4 ng mL-1 which is

361

substantially better than the traditional ELISA with an LOD of 93.8 ng mL-1. The obtained

362

results indicate that the AuNPs-LISA assay can detect low levels of OvAg utilizing the

363

peroxidase-like activity of the AuNP probe. The peroxidase-like activity of AuNP has been

364

successfully used to detect human IgG (H-IgG) as an analytical model for a naked-eye

365

sensitive ELISA-like assay with a low LOD [30]. Thus, the proposed AuNPs-LISA assay is

366

a feasible method for OvAg detection in urine samples. It should be noted that not only a

367

lower LOD can be obtained from the proposed AuNPs-LISA, but also that the operation time

368

is also reduced due to the omission of the incubation step of the conjugated-biotins as

369

compared to the traditional ELISA. Insert Fig. 5 here.

370

371

3.4 Field application of AuNPs-LISA for OvAg detection in urine samples

372

Further studies are required to evaluate the practical feasibility for the field

373

application of this method. Therefore, we tested the method using urine samples collected

374

from individuals in an endemic area for Ov. As shown in Table 1, the 390 samples were

375

separated into 130 from males and 260 from females. We found 54 cases Ov positive samples

376

in males (42%), the 76 female cases (29%). The mean age of the subject group was 53±11.47

377

years. We then separated the ages into 3 groups. There were 231 individuals aged more than

378

50 years, followed by 106 cases of 41-50 years old and the 53 cases of 31-40 years old. We

379

found that 89 Ov positive cases fell within the >50 years group (39%), while 33 cases were

380

Ov positive in the 41-50 years group (31% of its interval) and 8 cases were found in the 31-

381

40 years old group (15% of its interval).

382

Insert Table 1 here.

383

Insert Fig. 6 here.

384

ROC curve analysis was established to compare to the traditional ELISA method

385

and the AuNP assay, as show in Fig. 6. The AuNPs-LISA assay showed a high AUC of

386

0.997. This result indicates that the AuNPs-LISA assay is potentiality a method to correctly

387

classify Ov positive individuals (a True Positive) and Ov negative individuals (a True

388

Negative) at least as well as those determined by traditional ELISA. A diagnostic cut-off (OD

389

value as 0.639) was obtained from ROC curve analysis and the diagnostic sensitivity and

390

specificity values were 93.81 and 91.34%, respectively (Table 2). The accuracy value of

391

AuNPs-LISA assay was 92.05% compared to traditional ELISA method (Table 2). To further

392

characterize the assay performance the following were calculated: positive predictive value

393

(PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood

394

ratio (NLR), and disease prevalence (DP): 81.54%, 97.31%, 10.83, 0.07, and 28.98%,

395

respectively (Table 2). Therefore, we have determined that the AuNPs-LISA assay is an

396

effective method for the diagnosis Ov infection in urine samples for the control and

397

elimination of human opisthorchiasis. Insert Table 2 here.

398

399

4. Conclusions and future outlook

400

The AuNPs-LISA assay was successfully tested using an AuNPs probe instead of the

401

usual HRP system of signal enhancement of the traditional ELISA for OvAg detection in

402

urine. The peroxidase-like activity of the AuNPs probe was induced by utilizing gold

403

enhancing solution, a set concentration of the AuNPs probe and H2O2. In addition, oxidized

404

TMB could be stabilized by ATP, which increased the signal from the AuNPs-LISA assay.

405

This model could decrease the number of steps and the time required for the traditional

406

ELISA method. It exhibits high diagnosis sensitivity and specificity. The AuNPs-LISA assay

407

showed excellent potential for the diagnosis of OvAg in urine samples from an endemic area.

408

Therefore, AuNPs-LISA is an effective method for OvAg detection in urine for the control

409

and elimination of opisthorchiasis in the Mekong River Basin region of Southeast Asia.

410

Acknowledgements

411

This work was supported by Cholangiocarcinoma Screening and Care Program

412

(CASCAP), Cholangiocarcinoma Research Institute (CARI) Khon Kaen University (grant

413

number

414

(IRN62W0002). We thank Professor Trevor N. Petney for editing the MS via the Publication

415

Clinic KKU, Thailand.

416

Conflict of interest

417

CASCAP-23),

the

Thailand

Science

Research

and

Innovation

(TSRI)

The authors have declared that do not have any conflict of interest.

418

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Figure captions Fig. 1 Schematic representation of the traditional ELISA method using the HRP system as the enhancing signal and the new AuNPs-LISA assay using an AuNP probe. Fig. 2 (A.)UV-vis absorbance spectra of the signal enhancing system containing (1) AuNPs probe only, (2) AuNPs probe + TMB, (3) AuNPs probe +H2O2, (4) AuNPs probe + TMB + H2O2, (5) AuNPs probe + gold enhancing solution, (6) AuNPs probe + gold enhancing solution + TMB, (7) AuNPs probe + gold enhancing solution + H2O2, (8) AuNPs probe + gold enhancing solution + TMB + H2O2, (9) gold enhancing solution + TMB + H2O2, (10) TMB + H2O2. The yellow reaction color was particularly pronounced in system 8 with peroxidase-like activity of the AuNPs probe. (B.)

The

three independent photographs of the signal enhancing system to validate the reproducibility of the proposed assay. Fig. 3 (A.) OD value at A450 nm for the different AuNPs probe dilutions (1:480, 1:240, 1:120, 1:60, and 1:30 dilution) (B.) UV-vis absorbance spectra for the different AuNPs probe concentrations and the corresponding color change (C.) OD value at A450 nm in presence of different H2O2 concentrations (0.8, 1.5, 3, 6, and 12%) (D.) UV-vis absorbance spectra and the corresponding color change against the different H2O2 concentrations. Fig. 4 (A.) UV-vis absorbance spectra of the signal enhancing system of AuNPs-LISA (1) absence of the enhancing solution and ATP, (2) absence of ATP, (3) absence of the gold enhancing solution, and (4) in presence of both the enhancing solution and ATP. (B.) ∆OD value at A450 nm and the corresponding color change for the signal enhancing system in the presence of the different ATP concentrations (0.078, 0.156, 0.313, 0.626, 1.25, and 2.5 mM) in the AuNPs-LISA assay.

Fig. 5 OD value at A450 nm and the changed reaction color for LOD detection at the different OvAg concentrations (12000, 6000, 3000, 1500, 750, 375, 187.5, 93.8, 46.9, 23.4, 11.7, and 5.9 ng mL-1) of (A.) the AuNPs-LISA assay was 23.4 ng mL-1 and (B.) traditional ELISA method was 93.8 ng mL-1. Fig. 6 Receiver operating characteristic (ROC) curves comparing the AuNPs-LISA assay to the traditional ELISA method (n = 390). Area under the curve of this model was 0.977.

Table 1 Characteristics of samples from the endemic area in the Lao People's Democratic Republic (n= 390) Sex N

390

Age

Male

Female

(Ov positive, %)

(Ov positive, %)

130 (54, 41.54)

260 (76, 29.23)

Mean±SD

53±11.47

Interval of Age 31-40

41-50

>50

(Ov positive, %)

(Ov positive, %)

(Ov positive, %)

53 (8, 15.09)

106 (33, 31.31)

231 (89, 38.53)

Table 2 Diagnostic performance of OvAg detection by the AuNPs-LISA assay compared to the traditional ELISA method in field-collected samples from the Lao People's Democratic Republic (n = 390). Diagnosis performance of AuNPs-LISA Comparator

AOC

cut-off

Traditional ELISA

0.977

0.639

*Note

PPV, Positive Predictive Value NLR, Negative Likelihood Ratio

Sensitivity

Specificity

Accuracy

PPV*

NPV*

(%)

(%)

(%)

(%)

(%)

93.81

91.34

92.05

81.54

97.31

NPV, Negative Predictive Value DP, Disease prevalence

PLR, Positive Likelihood Ratio

PLR*

NLR*

10.83

0.07

DP* (%) 28.98

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Highlights: •

The enzyme system of the traditional ELISA was replaced by an AuNPs probe

•

The gold enhancing solution and the addition of ATP can improve the peroxidase-like activity of the AuNPs probe

•

The proposed AuNPs-LISA can detect a low level for OvAg

•

AuNPs-LISA has high sensitivity and specificity to detect OvAg in urine

•

AuNPs-LISA can be used in the diagnosis of opisthorchiasis in real urine samples

Conflict of interest The authors have declared that do not have any conflict of interest.