Novel Strategies To Enhance Lateral Flow Immunoassay Sensitivity

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Novel Strategies to Enhance Lateral Flow Immunoassay Sensitivity for Detecting Foodborne Pathogens: A Review Weihua Lai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5046415 • Publication Date (Web): 24 Dec 2014 Downloaded from http://pubs.acs.org on December 30, 2014

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Novel Strategies to Enhance Lateral Flow Immunoassay Sensitivity for Detecting Foodborne Pathogens: A Review

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Journal of Agricultural and Food Chemistry jf-2014-046415.R1 Review 23-Dec-2014 Shan, Shan; State key Laboratory of Food Science and Technology, Lai, Weihua; State Key Laboratory of Food Science and Technology, Nanchang University, Xiong, Yonghua; 2Jiangxi-OAI Joint Research Institute, Nanchang University, Wei, Hua; State Key Laboratory of Food Science and Technology, Nanchang University, Xu, Hengyi; State Key Laboratory of Food Science and Technology, Nanchang University,

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Novel Strategies to Enhance Lateral Flow Immunoassay Sensitivity

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for Detecting Foodborne Pathogens: A Review

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Shan Shan, Weihua Lai*, Yonghua Xiong, Hua Wei, Hengyi Xu

4

State Key Laboratory of Food Science and Technology, Nanchang University,

5

Nanchang 330047, China

6 7 8 9 10 11 12 13 14

*Send correspondence to:

15

Weihua Lai, Ph.D, Professor

16

State Key Laboratory of Food Science and Technology, Nanchang University

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Address: 235 Nanjing East Road, Nanchang 330047, China

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Tel: 0086-13879178802; Fax: 0086-791-88157619;

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E-mail: [email protected]

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Abstract: Food contaminated by foodborne pathogens causes diseases, affects

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individuals, and even kills these affected individuals. As such, rapid and sensitive

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detection methods should be developed to screen pathogens in food. One of current

24

detection methods is lateral flow immunoassay, an efficient technique because of

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several advantages, including rapidity, simplicity, stability, portability, and sensitivity.

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In this review, we present the format and principle of lateral flow immunoassay strip

27

and the development of conventional lateral flow immunoassay for detecting

28

foodborne pathogens. Further, we focus on novel strategies that can be applied to

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enhance the sensitivity of lateral flow immunoassay to detect foodborne pathogens;

30

these strategies include innovating new labels application, designing new formats of

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lateral flow immunoassay, combining with other methods, and developing

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signal-amplification systems. With these advancements, detection sensitivity and

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detection time can be greatly improved.

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Key words: lateral flow immunoassay, foodborne pathogens, sensitivity enhancement

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1. Introduction

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Food safety is an important public health priority. Foodborne diseases are serious

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global problems and impose a significant burden not only on infected individuals but

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also on the economy. Although a majority of cases are mild, occasional cases can lead

40

to serious or long-term conditions or even death. The Centers for Disease Control and

41

Prevention estimated that each year roughly one in six Americans (or 48 million

42

people) became adversely affected by foodborne diseases.1 The World Health

43

Organization estimated that approximately a million people suffered from foodborne

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diseases each year in the UK.2 Foodborne pathogens contaminate food at some parts

45

of a food chain from farm to fork. Salmonella, enterohemorrhagic Escherichia coli

46

(EHEC),

47

Cronobactor, Vibrio, and Shigella are reported frequently. For instance, Salmonella is

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one of the most common causes of food poisoning in the world and can cause more

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serious illnesses in older adults, infants, and persons with chronic diseases. Every year,

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Salmonella is estimated to cause approximately 1.2 million illnesses in the United

51

States.3 Another case involves EHEC; this pathogen is often reported as one of the

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most common causes of foodborne outbreaks. For example, approximately 265,000

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EHEC infections occur each year in the United States. EHEC O157 also causes

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approximately 36% of these infections, and non-O157 EHEC accounts for the

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remaining cases.4 In other instances, L. monocytogenes primarily affects older adults,

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pregnant women, newborns, and adults with weakened immune systems. In the

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United States, L. monocytogenes causes nearly 2,500 cases of listeriosis per year.5

Listeria

monocytogenes,

Staphylococcus

aureus,

Campylobacter,

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Foods that are most frequently associated with foodborne illnesses include meat, fish,

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poultry, vegetables, and fruits. These foods may cause illnesses when they are eaten.

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In traditional method, analytes are initially cultured in enrichment media for 18 h

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to 24 h and then cultured in selective media for 18 h to 24 h; biochemical and

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serological tests are subsequently performed to confirm suspected colonies for another

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3 d to 5 d. Conventional detection methods require 5 d to 7 d to identify target

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bacteria. The methods of conventional isolation and culture are time-consuming and

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laborious. Therefore, a simple, specific, and sensitive method that can be used to

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detect pathogens should be developed. Several methods based on nucleic detection,

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such as real-time polymerase chain reaction (PCR)6-8, multiplex PCR9-11, and

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loop-mediated isothermal amplification (LAMP) method12-14, have been developed

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rapidly. Enzyme-linked immunosorbent assay (ELISA)15-17, biosensing18-21, and

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electrochemical strategies22-23 have also been extensively investigated. On the one

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hand, these methods are sensitive and specific; on the other hand, these methods

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require technical expertise, laborious procedures, and expensive instrumentation. In

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addition, these technologies are inappropriate for on-site tests. With these drawbacks,

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detection techniques have been developed and improved. For instance, lateral flow

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immunoassay (LFI) has been considered as a promising diagnostic tool extensively

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applied to detect drugs24-27, toxins28,29, hormones30,31, heavy metals32,33, pesticides34,35,

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and pathogens36,37. LFI provides several advantages, including rapid procedure,

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convenient operational requirements, sensitivity, and cost-effective equipment.

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Review papers have described the strengths, weaknesses, opportunities, and threats of

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LFI38, as well as its advanced ability to detect mycotoxins, phycotoxins, and

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pesticides39,40. In this paper, recent advances on LFI applied to detect foodborne

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pathogens are described. Furthermore, sensitivity enhancement methods are presented

83

and discussed.

84 85

2. Format and principle of traditional LFI

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2.1 Components of the LFI format

87

Immunochromatography test paper technology, which was developed in the early

88

1980s, involves rapid detection based on immunological techniques. A LFI device

89

consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an

90

absorbent pad. A test zone and a control zone are found on the nitrocellulose

91

membrane (Fig. 1A).

92 93

Sample pads are used to filter specific sample components; these pads can also change the pH of a sample and release analytes with high efficiency.

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Conjugate pads are used to accept recognition labels and keep these labels stable

95

during their entire shelf-life period. Recognition labels are added to a treated

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conjugate pad by impregnating them in a conjugate suspension or dispensing them

97

with quantitative non-contact dispensers.

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After a sample is added to a strip, this sample migrates through a sample pad to a

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conjugate pad, where recognition labels are immobilized. Analytes in a given sample

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then interact with recognition labels when they migrate to an analytical region.

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Specific biological components are then bound at a test line and a control line via

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quantitative dispensers. Absorbent pad resembles an engine of an LFI device; absorbent pad pulls fluid

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and retains this fluid during the whole assay.

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2.2 LFI principle to detect foodborne pathogens

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Competitive assay and sandwich assay are the two kinds of formats commonly

107

used in LFI. Competitive assay is preferred when target analytes exhibit low

108

molecular weight or present single specific antigen. Sandwich assay is performed

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when target analytes present several epitopes, such as pathogens. In a sandwich assay,

110

the first analyte ligand (monoclonal antibodies are commonly used) is used to label

111

markers. The second ligand (polyclonal antibodies are widely used) is placed on the

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test zone of a nitrocellulose membrane and assigned as the test line. The specific

113

antibodies coating the control line capture excess labeled antibodies and conjugates.

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As sample extract is applied to the LFI device, the labeled first ligand initially binds

115

to the target analyte to form an analyte-ligand complex; this complex then migrates

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forward by capillary force to the test zone. The second ligand in the test zone

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subsequently binds to the target analyte. Detectable signal enhancement in the test

118

zone directly corresponds to the amount of a target analyte in a sample. In sandwich

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assay, detectable signal enhancement is observed as target analyte concentration

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increases.

121 122 123

3. Conventional LFI to detect foodborne pathogens Colloidal gold lateral flow strip is considered as one of the most widely used

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tools to detect foodborne pathogens. Colloidal gold is essentially inert and forms

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spherical particle. Antibodies bind to the surfaces of these gold particles with

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enormous strength when correctly coupled, thus providing a high degree of long-term

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stability.41,42 Hence, ligands exhibit good compatibility with biomolecules and can

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retain biochemical activities of tagged biomolecules. Colloidal gold-based LFI has

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been applied to detect foodborne pathogens.43-50 Furthermore, a LFI device has been

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used to determine the analytical sensitivity of E. coli O157 of approximately 105

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CFU/mL51,52. Preechakasedkit et al.53 also used colloidal gold-based LFI strip to

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detect S. typhimurium, and the limit of detection was 1.14 × 105 CFU/mL. Ueda et

133

al.54 applied the colloidal gold LFI strip to detect L. monocytogenes, and the detection

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limit of this assay was 106 CFU/mL. The detection limit of Vibrio harveyi was 106

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CFU/mL when a colloidal gold LFI was used.55 Wiriyachaiporn et al.56 further

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developed an LFI device to detect Staphylococcus aureus, and the result showed a

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detection limit of 106 CFU/mL. Colloidal gold-based LFI has also been widely

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applied in commercial detection. Table 1 summarizes the information regarding

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conventional colloidal gold LFI applied to detect foodborne pathogens in industries

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from some manufacturers. Although this summary includes only several commercial

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products, the main trends of colloidal gold LFI in the detection of foodborne

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pathogens are shown.

143 144 145

4. Recent advancements in LFI to detect foodborne pathogens Researchers have devoted efforts to develop a rapid and sensitive lateral flow

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assay platform to screen foodborne pathogens (Table 2). New advancements in lateral

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flow assay have also been developed to detect foodborne pathogens; these

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advancements include innovating novel labels, designing new formats of LFI,

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combining with other methods, and developing signal-amplification systems.

150

4.1 Innovation of novel label application

151

An ideal label should be stable, low non-specific binding, cost effective, and

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easy for conjugation; an ideal label could also be used for multi-analyte detection.

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Many efforts have also been devoted to discover novel labels to enhance detection

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sensitivity. In this paper, the types of the new labels successfully applied to enhance

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the sensitivity of LFI to detect foodborne pathogens were described; these new labels

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include colored labels, luminescent nanoparticles (NPs), and labels based on other

157

properties.

158

4.1.1 Colored labels

159

Colored labels are visible to the naked eyes. They have been widely used in a lot

160

of fields owing to their convenience, such as colloid gold and colored latexes are the

161

most common labels in LFI. Carbon nanoparticles have also been applied as labels

162

owing to their low price, nontoxicity and high signal intensity, and they can easily

163

bind ligands to their large surface area, and they are suitable for LFI detection because

164

of their “black-on-white” test results.57,58

165

Blažková59 used carbon as a label to develop a nucleic acid test strip to detect

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Cronobacter spp. The PCR products of the analytes were conjugated with biotin on

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one side and digoxigenin on the other side. Carbon NPs were labeled with neutravidin

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as capture conjugate. Anti-digoxigenin antibodies and biotin-protein conjugates were

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sprayed on NC membrane as test line and control line, respectively. The detection

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limit of PCR product was 8 ng.

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4.1.2 Luminescent NPs

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Luminescent NPs have been extensively investigated because these NPs exhibit

173

high sensitivity and spectral characteristics. Fluorescent dyes60, quantum dots61, and

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up-converting emitters62 have been applied as labels to detect Campylobacter jejuni, E.

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coli O157:H7, and Vibrio in an LFI system.

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Fluorescent dyes display low photo-stability and are highly quenched by the

177

environment and concentration63. To overcome these drawbacks, researchers should

178

couple multiple fluorescent dyes to a carrier or produce particles loaded with

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fluorescent dyes. Xie et al.64 applied LFI based on fluorescent microspheres as labels

180

to detect E. coli O157:H7 with the reader which was from Shanghai Huguo Science

181

Instrument Co, Ltd (excitation wavelength = 470 nm; emission wavelength = 520 nm).

182

The results showed that the sensitivity of this LFI system was 104 CFU/mL while the

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sensitivity of LFI based on colloidal gold was 105 CFU/mL.

184

Quantum dots have been applied to bioassays and biosensors due to excellent

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signal brightness, size-tunable light emission, broad excitation spectrum, narrow

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emission spectrum, and excellent stability resisting photobleaching.65-67 Bruno68

187

compared colloidal gold with quantum dot versions in lateral flow test strips to detect

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E. coli O157:H7. A handheld long wave UV light (365 nm) and an orange Schott

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glass filter were used for illumination and enhancing visual detection of quantum dot

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particles. The limit of detection of E. coli O157:H7 were 6,000 and 600 cells per test

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in a buffer by using colloidal gold lateral flow assay system and quantum dot lateral

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flow assay system, respectively.

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Up-converting emitters are reported more sensitive than conventional labels such

194

as colloidal gold or colored latex beads.69 Up-converting emitters exhibit several

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advantages, including long lifetime and sequential two-photon absorption possible in

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a microsecond time scale. With these emitters, background absorption can be

197

minimized because of anti-stokes luminescence, and biomolecule photodegradation

198

does not occur because of excitation occurring in the infrared area and the ease of

199

production.70 An ultrasensitive up-conversion fluorescent lateral flow strip based on

200

NaYF4:Yb,Er NPs was applied to detect Vibrio anguillarum.62 Carboxyl-modified

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β-NaYF4:Yb,Er NPs were synthesized by a facile one-pot solvothermal approach and

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then coupled with mAb as a conjugate probe in lateral flow assay. Continuous wave

203

near infrared spectrum laser at λ = 980 nm was used to excite the upconversion

204

nanoparticles on the strip by the Up-converting nanoparticles-LFI biosensor. The

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sensitivity of the Up-converting fluorescent strip was 102 CFU/mL and 100 times

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higher than that in enzyme-linked immunosorbent assays, exhibited good specificity

207

attributed to no cross reaction with eight other pathogens.

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4.1.3 Other labels

209 210 211

In addition to colored and luminescent particles, new labels, including magnetic beads, enzymes, and liposomes, are also applied in LFI. Magnetic beads have been widely used to separate target analytes; these

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magnetic beads have also been used as carriers and labels to detect pathogens.71-74

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Wang et al.75 developed a super-paramagnetic lateral flow assay to detect Bacillus

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anthracis spores.

215

super-paramagnetic beads to capture bacterial spores. Different anti-B. anthracis

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mAbs were sprayed onto a nitrocellulose membrane as a test line, and goat anti-mouse

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IgG was used as a control line. The detection system yielded a linear range of 4 × 103

218

CFU/mL to 4 × 106 CFU/mL, and reproducible detection limits were 200 spores mg–1

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in milk powder. These super-paramagnetic beads were larger and reaction time was

220

longer than other types of strip tests because spores were larger (approximately 1 µm

221

in diameter) than other analytes (such as proteins). The conjugates that run through a

222

membrane required a long reaction time when the conjugates of magnetic beads and

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antibodies captured the spores. Small-diameter particles were also used as labels.

224

With these particles, flow rate was higher and reaction time was shorter; however, the

225

use of these materials reduced detection sensitivity. Yan et al.76 discussed the effect of

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the characteristics of Fe3O4 NPs (including particle size, size distribution, surface

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biomodification, magnetic property, and colloidal stability) on LFI when these Fe3O4

228

NPs were used as labels to detect V. parahaemolyticus. To increase sensitivity and

229

decrease false positive reaction, they chose Fe3O4 NPs which had small size and good

230

colloidal stability as the labels. The 200nm Fe3O4 nanoparticles were selected for the

231

detection system.

Anti-B.

anthracis

mAbs

were

conjugated

to

300

nm

232

In another approach, enzymes as labels were introduced to lateral flow assay to

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decrease the limit of detection. Enzymes can be linked to biomolecules to form

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complexes and exhibit enzymatic and immunological activities. The reaction between

235

enzymes and substrates amplifies optical signals. Enzyme-linked immunosorbent

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assay (ELISA) based on the reaction between enzymes and substrates are considered

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as one of the most outstanding assays. Hence, ELISA combined with lateral flow

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assay to detect analytes could be an excellent strategy to amplify signals.77 Park et

239

al.78 fabricated an LFI based on sandwich ELISA to detect E. coli O157:H7. Optical

240

signals were generated by the reaction between horseradish peroxidase (HRP) and its

241

substrate, which could be read directly by naked eyes. The lower limit of detection

242

was 103 CFU/mL.78 A similar method was applied to detect S. typhimurium. An

243

immunostrip was used to quantitatively determine S. typhimurium in the range of 9.2

244

× 103 CFU/mL to 9.2 × 106 CFU/mL based on chemiluminescent signals.79

245

Liposomes are formed from amphiphilic molecules and can encapsulate and

246

immobilize molecular markers, such as dyes, via covalent or hydrophobic interactions.

247

Khreich et al.80 encapsulated sulfordamine B in liposomes as labels to detect

248

fluorescent signals and obtained a 15-fold increase in sensitivity compared with

249

optical detection sensitivity of colored labels. Immunoliposome-encapsulated

250

sulfordamine B has also been applied as a capture label to detect S. typhimurium and

251

compared with traditional colloidal gold lateral flow strip.81-83 The result showed that

252

the sensitivity of immunoliposome immunoassay strip was higher than that of

253

traditional colloidal gold immunoassay strip; this high sensitivity was appropriate to

254

detect food matrix.

255

4.2 New formats of LFI

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The application of antibodies in a LFI system exhibits several weaknesses,

257

including difficult preparation and poor stability. A new ligand should be discovered

258

to replace antibody. With advances in nucleic acid- and genomic sequence-based

259

techniques, various detection methods have been developed using nucleic acid rather

260

than antibodies. A rapid and sensitive detection system, which is called nucleic acid

261

LFI84-86, has been developed. In the detection of S. aureus by nucleic acid LFI,

262

quantum dot-streptavidin conjugate was used as a capture probe. Anti-digoxigenin

263

antibody and BSA-biotin conjugate were sprayed on a membrane as a control line and

264

a test line, respectively.87 This detection system was based on two reaction systems,

265

including digoxigenin and antidigoxigenin, streptavidin and biotin. DNA extracted

266

from the analytes in a sample was labeled with digoxigenin on one side and with

267

biotin on the other side. The schematic of detection principles was shown in Fig. 1B.

268

The limits of detection of S. aureus were 3 CFU/mL and 30 CFU/g in spiked milk

269

powder and meat samples, respectively. Extracted DNA from analytes in a sample is

270

usually amplified by PCR. LAMP was applied to perform rapid and sensitive nucleic

271

acid amplification combined with lateral flow assay.88-90 A shorter time is needed to

272

amplify target DNA by LAMP than by PCR because a set of four primers that

273

recognize a total of six distinct sequences on the target DNA is used in LAMP; LAMP

274

also relies on the auto-cycling strand displacement of DNA synthesis performed by

275

the large DNA polymerase under isothermal conditions.

276

Aptamers are another choice to replace antibodies as recognition molecules used

277

for bacterial detection methods to bind specific analytes.91-94 Yonekita et al.95 used

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antimicrobial peptides (AMPs) as recognition probes for bacteria in a LFI.

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Streptavidin-colloidal gold was conjugated with biotinylated AMP, which was used as

280

a capture probe. Anti-analyte antibodies were then sprayed on a test line while

281

anti-streptavidin antibodies were sprayed on a control line. Moreover, LFI was

282

applied to simultaneously detect and identify Shiga toxin-producing E. coli O157,

283

O26, and O111 (Fig. 3). Studies have focused on the simultaneous detection by

284

LFI.96-97 Simultaneous detection system exhibits several advantages, such as simple,

285

rapid, and sample-saving, compared with single detection. Yu et al.98 developed a

286

colloidal gold LFI to simultaneously detect V. cholerae serogroups O1 and O139,

287

which could cause epidemic and pandemic cholera. The result showed that 100%

288

sensitivity and 100% specificity were obtained for O1 and O139 in all of the tested

289

strains. The loss of intensity in the test line after 21 weeks was not significant

290

compared with that after 4 weeks; LFI remained stable when stored dry at room

291

temperature for at least three months.

292

4.3 Combined with other methods

293

The sensitivity and specificity of methods used to detect foodborne pathogens

294

should be improved. Immunomagnetic separation (IMS) performs an increasingly

295

important function in the isolation and concentration of foodborne pathogens in food

296

sample.99-101 A detection method of combined IMS and LFI can significantly improve

297

sensitivity, decrease detection time, and eliminate interference from food

298

matrices.102-104 Cui et al.105 used IMS and traditional colloidal gold LFI to detect E.

299

coli O157:H7. The results showed that the detection limit was 7.6 × 103 CFU/mL;

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thus, the result was 10 times higher than that of colloidal gold lateral flow assay. The

301

schematic was shown in Fig. 3. Chen et al.106 developed an LFI test to detect

302

Cronobacter and used silica-coated magnetic NPs to separate nucleic acid from

303

Cronobacter lysate. The detection limit of this technique was 107 CFU/mL in pure

304

culture, and 1 mL of 105 CFU Cronobacter was detected as positive one in pure

305

culture after silica-coated magnetic NP treatment was administered.

306

In a food matrix, some non-target analytes may cause false-positive results. In

307

the primary enrichment phase, the growth of closely related non-target bacteria should

308

be suppressed to improve detection sensitivity. Muldoon and co-workers107 applied

309

specific bacteriophages, which could suppress non-Salmonella bacterial growth for

310

the control of cross-reactive non-Salmonella bacteria during primary sample

311

enrichment. The bacteriophage cocktail that was added into primary enrichment

312

significantly reduced the false positives of immunochromatographic test strip. False

313

positives were reduced from 32 of 115 samples tested to zero in naturally

314

contaminated beef samples.

315

4.4 Development of the signal-amplification system

316

4.4.1 Application of a biotin-streptavidin system

317

Several approaches have been used to amplify signals of traditional gold

318

colloidal LFI. These approaches aim to improve LFI sensitivity. To increase

319

sensitivity effectively, a biotin-streptavidin system has been applied in lateral flow

320

assay. Biotin-streptavidin system exhibits good reaction specificity and high affinity;

321

hence, this system has been widely applied in many field detections.108,109

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Furthermore, this system can greatly contribute to signal amplification and

323

background

324

immunochromatographic strip based on a biotin-streptavidin system to detect E. coli

325

O157:H7. Colloidal gold labeled anti-E. coli O157 monoclonal antibodies and

326

biotinylated anti-E. coli O157 polyclonal antibodies were impregnated in a conjugated

327

pad and a sample pad, respectively. Streptavidin and goat anti-mouse antibodies were

328

immobilized in a test line and a control line of nitrocellulose membrane, respectively.

329

E. coli O157 bound to biotinylated antibody and colloidal gold-labeled antibody to

330

form a biotinylated antibody-analyte-colloidal gold-labeled antibody complex. This

331

complex migrated to the detection zone by capillary action. The test line became red

332

when the complex was captured by streptavidin via high-affinity biotin-streptavidin

333

interaction (Fig. 4). Streptavidin coated in the test line could reduce the background

334

signal and amplify detection signal owing to the high affinity between biotin and

335

streptavidin. This method could detect E. coli O157 at a minimum of 2.3 × 103

336

CFU/mL without enrichment. Sensitivity was near 100 times higher than that of

337

traditional lateral flow assay.52

338

4.4.2 Enzyme amplification

activity

reduction.110

Zhao

et

al.111

prepared

colloidal

gold

339

In another approach involving signal amplification, enzymes are introduced to

340

lateral flow assay. Cho et al.112 prepared an antibody-enzyme-magnetic NP complex

341

as a label to detect L. monocytogenes by employing lateral flow assay. In this strategy,

342

magnetic properties and enzyme catalytic amplifications were combined, thereby

343

generating a darker signal on magnetic beads because of enzyme-substrate reaction

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(Fig. 5). The study included three steps as follows: 1) magnetic separation and

345

concentration; 2) lateral-flow immunochromatography; and 3) signal generation by

346

placing HRP substrates on the absorption pad on the lateral side of a nitrocellulose

347

membrane pad (Fig. 5). Signals were captured as images using a scanner. The optical

348

densities of the images were digitized by using image analysis program (Multianalyst

349

version 1.1, Bio-Rad Laboratories, Hercules, CA) to obtain the signal value. After

350

signal was amplified, the limit of detection of 95 CFU/mL was obtained in buffer

351

solution.

352

4.4.3 Silver enhancement

353

Silver nucleation on gold has been applied to perform sensitive biosensing and

354

achieve signal amplification.113-116 Silver precipitates on gold and can enhance

355

electrochemical and colorimetric signals; silver can also vary the resistance or

356

conductance of a substrate.117 Color is greatly enhanced on the basis of the reaction

357

between silver salt and reducing agent when silver is deposited on gold. Liu et al.118

358

utilized a 16S rDNA/rRNA probe-conjugated AuNPs and silver enhancement method

359

based on sandwich LFI to detect Salmonella. In this assay, anti-avidin antibody was

360

sprayed onto a nitrocellulose membrane to capture avidin. A biotinylated capture

361

probe was conjugated with avidin and 16S rDNA/rRNA probe-conjugated AuNPs as a

362

capture probe to capture Salmonella-targeted nucleic acid. Both biotinylated probe

363

and probe-conjugated AuNPs could capture Salmonella-targeted nucleic acid; these

364

probes then constituted sandwich LFI. To improve detection sensitivity and intensify

365

AuNP signal, researchers soaked test strips in silver enhancer reagents. Nucleic acids

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were detected from 107 bacterial cells. The detection limit was as low as 104 cells

367

after silver enhancement was performed (Fig. 6).

368 369

5. Conclusions

370

Traditional lateral flow assay has been widely applied to detect foodborne

371

pathogens. However, detection sensitivity is limited. As such, future studies should

372

focus on enhancing detection sensitivity and decreasing detection time of LFI to

373

detect foodborne pathogens. Scientists have devoted efforts to attain these goals. This

374

review described recent LFI advancements, including using new labels, applying new

375

formats, combining other methods, and utilizing signal-amplification systems, in

376

foodborne pathogen detection. With these advancements, detection sensitivity and

377

detection time can be greatly improved. In the future, hard word should be done to

378

further improve the stability and convenience of these sensitive lateral flow

379

immunoassays.

380 381

Acknowledgements

382

We are grateful to the Research Program of State Key Laboratory of Food Science

383

and Technology, Nanchang University (Project No. SKLF-ZZB-201307), earmarked

384

fund for Jiangxi Agriculture Research System (JXARS-03), and the Nanchang

385

Technological Program (2012-CYH-DW-SP-001) for financial support.

386

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Ahmed,

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Anfossi, L.; Di, N.F.; Giovannoli, C.; Passini, C.; Baggiani, C. Increased

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sensitivity of lateral flow immunoassay for ochratoxin A through silver

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779

immunochromatographic assay for the rapid detection of abrin-a. Biosens

780

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Journal of Agricultural and Food Chemistry

783 784 785 786

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788

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789

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790

Figure Captions

791

Figure 1 Components of LFI strip format (A). Schematic of detecting S. aureus

792

principles of nucleic acid LFI (B) (Adapted from Chen et al87).

793 794

Figure 2 Schematic of the multiplex LFI strip. Anti-analytes antibodies were sprayed

795

on test lines to capture E. coli O157 (A), E. coli O26 (B), and E. coli O111 (C). Analytes

796

were mixed with streptavidin-colloidal gold conjugate solution, and the mixtures were

797

added to the sample pad of the LFI strip. After 15 min, positive or negative results

798

could be determined (Adapted from Yonekita et al95).

799 800

Figure 3 Schematic of immunomagnetic separation combined with colloidal gold LFI

801

(Adapted from Cui et al105).

802 803

Figure 4 Schematic of the colloidal gold LFI based on biotin-streptavidin system to

804

detect E. coli O157:H7

805 806

Figure 5 Schematic of LFI based on enzyme combined with magnetic separation step

807

to detect L. monocytogenes (Adapted from Cho et al112)

808 809

Figure 6 Schematic of LFI. As immunoreaction was completed, test strip was soaked

810

in silver enhancer reagent to intensify the signal of gold nanoparticles (Adapted from

811

Liu et al 118).

812 38

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813

Tables

814

Table 1 Commercial colloidal gold LFI applied to detect foodborne pathogens Company

Web site

Analytes

www.kwinbon.com

E. coli O157:H7, Listeria monocytogens

Biocontrol

www.biocontrolsys.com

EHEC, Salmonella, Listeria monocytogens

DuPont

www.dupont.com

E.

Beijing

Kwinbon

Biotechnology

coli

O157:H7,

Salmonella,

Listeria

monocytogens Invisible sentinel

invisiblesentinel.com

E.

coli

O157:H7,

STEC,

Salmonella,

Listeria .spp, Campylobacter Merck KGaA/EMD

www.merck.com

E. coli O157:H7, Salmonella,Campylobacter

www.neogen.com

E.

Chemicals Neogen company

coli

O157:H7,

Salmonella,

Listeria

monocytogens New

Horizons

www.nhdiag.com

E. coli O157:H7, Salmonella.spp

www.nipponham.co.jp

E.

Diagnostic Nippon

Meat

Packers Quicking Biotech

coli

O157,

O26,

O111,

Salmonella

enteritidis, Listeria .spp, Campylobacter jejuni www.quicking.cn

E.

coli

O157:H7,

Salmonella,

Listeria

Salmonella,

Listeria

Salmonella,

Listeria,

monocytogens Romer labs

www.romerlabs.com

E.

coli

O157:H7,

monocytogens Ubio Biotechnology www.ubio.in

E.

coli

O157:H7,

System

Shigella, Vibrio cholera, V. parahemolyticus

815 816

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817 818

Page 40 of 47

Table 2 Novel strategies applied in LFI for detecting foodborne pathogens Methods enhance detection sensitivity Colored Labels

819 New label application

Luminescent NPs

Other Labels New formats of LFI Combined with other methods Signal-amplification system

Nucleic Acid LFI AMPs LFI&Simultaneous detection Combined with IMS Biotin-streptavidin system Enzyme amplification Silver enhancement

Target Shiga toxin-producing E. coli Cronobacter spp E. coli O157:H7 E. coli O157:H7 V. anguillarum B. anthracis E. coli O157:H7 S. typhimurium S. aureus Shiga toxin-producing E. coli E. coli O157:H7 E. coli O157:H7 L. monocytogenes Salmonella

Limit of detection 4

5

10 /10 CFU/mL 8 ng 104 CFU/mL 6×102 CFU/mL 102 CFU/mL 200 spores/mg 103 CFU/mL 102 CFU/mL 3 CFU/mL 104 CFU/mL 7.6×103 CFU/mL 2.3×103 CFU/mL 95 CFU/mL 104 CFU/mL

Ref 58 59 64 68 62 75 78 81 87 95 105 111 112 118

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820

Figures

821

822 823

Figure 1

824

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825

826 827

Figure 2

828

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Journal of Agricultural and Food Chemistry

829

830 831

Figure 3

832

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833

834 835

Figure 4

836

44

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Journal of Agricultural and Food Chemistry

837

838 839

Figure 5

840

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841

842 843

Figure 6

844

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Journal of Agricultural and Food Chemistry

845 846

TOC graphic

847

The novel strategies can be applied in LFI to improve sensitivity. The labels of LFI include

848

colored particles, fluorescent microspheres, quantum dots, up-converting nanoparticles, magnetic

849

beads, enzymes, and liposomes. New formats of LFI include selecting DNA/RNA or aptamer as

850

the ligands, simultaneously detecting many kinds of analytes. The immunomagnetic

851

separation-LFI system and the signal-amplification systems including biotin-streptavidin system,

852

enzyme amplification, and sliver enhancement are also applied recently.

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