Aptamer-Based Paper Strip Sensor for Detecting - ACS Publications

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Aptamer based paper strip sensor for detecting Vibrio fischeri Woo-Ri Shin, Simranjeet Singh Sekhon, Sung-Keun Rhee, Jung Ho Ko, Ji-Young Ahn, Jiho Min, and Yang-Hoon Kim ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00190 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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ACS Combinatorial Science

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Graphical Abstract

Aptamer based paper strip sensor for detecting Vibrio fischeri

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Woo-Ri Shin1, Simranjeet Singh Sekhon1, Sung-Keun Rhee1, Jung Ho Ko2,

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Ji-Young Ahn1*, Jiho Min3* and Yang-Hoon Kim1*

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1

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School of Biological Sciences, Chungbuk National University 1 Chungdae-Ro, Seowon-Gu, Cheongju 28644, South Korea

College of Veterinary Medicine, Western University of Health Sciences, 309 E Second Street, Pomona CA 91766, USA 3

Department of Bioprocess Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, Jeonbuk 54896, South Korea

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[TOC graphic]

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*Correspondence should be addressed to Ji-Young Ahn (Phone) +82-43-261-2301, (Fax) +82-

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43-264-9600, (E-mail) [email protected] or Jiho Min (Phone) +82-63-270-2436, (Fax)

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+82-63-270-2306, (E-mail) [email protected] or Yang-Hoon Kim (Phone) +82-43-261-3575,

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(Fax) +82-43-264-9600, (E-mail) [email protected]

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Manuscript Submitted to ACS Combinatorial Science

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ACS Paragon Plus Environment

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Aptamer Based Paper Strip Sensor for Detecting Vibrio fischeri

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Woo-Ri Shin1, Simranjeet Singh Sekhon1, Sung-Keun Rhee1, Jung Ho Ko2,

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Ji-Young Ahn1*, Jiho Min3* and Yang-Hoon Kim1*

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School of Biological Sciences, Chungbuk National University 1 Chungdae-Ro, Seowon-Gu, Cheongju 28644, South Korea

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College of Veterinary Medicine, Western University of Health Sciences, 309 E Second Street, Pomona CA 91766, USA

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Department of Bioprocess Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, Jeonbuk 54896, South Korea

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Abstract: Aptamer-based paper strip sensor for detecting Vibrio fischeri was developed. Our

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method was based on the aptamer sandwich assay between whole live cells, V. fischeri and DNA

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aptamer probes. Following 9 rounds of Cell-SELEX and one of the negative-SELEX, V. fischeri

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Cell Aptamer (VFCA)-02 and -03 were isolated, with the former showing approximately 10-fold

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greater avidity (in the sub-nanomolar range) for the target cells when arrayed on a surface. The

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colorimetric response of a paper sensor based on VFCA-02 was linear in the range of 4 × 101 to

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4 × 105 CFU/mL of target cell by using scanning reader. The linear regression correlation

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coefficient (R2) was 0.9809. This system shows promise for use in aptamer-conjugated gold

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nanoparticle probes in paper strip format for in-field detection of marine bioindicating bacteria.

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Keywords: Vibrio fischeri; ssDNA Aptamer; Aptamer-based paper strip sensor;

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ACS Paragon Plus Environment 2

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Introduction

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Recent studies related to marine pathogen and public health are addressing a range of

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issues in marine environmental toxic assessment 1, 2. Biological polluted sea-water involved

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large quantities of density microbial in the form of biofilm, flocs or granules

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analytical detection methods have been applied, that not only represents a comparison of

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different approaches but also take into consideration the standardization of sample collection

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and processing methods

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bioindicator for aquatic toxic assessment. Marine bacteria as a bioindicator should be the

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high variability of microbial communities observed naturally and detect environmental

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changes due to the presence of pollutants, as well as species present in it.

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Vibrio fischeri is a strain of Vibrio that grows on the light emitting organs of deep-sea squid 8,

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3, 4, 5

. New

6, 7

. However, it is required to develop universally accepted

9

. It is a tuberous anaerobic gram-negative bacterium that is motile and has one or more

72

flagella. V. fischeri has been found globally in marine environments and served as a model

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organism to understand the bioluminescent regulation and biofilm formation as a strategy to

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increase viability

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compared to the non-trivial group and is an essential step in survival strategy 13. Additionally,

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the biofilm formation may have adverse effects on the economy and industry even if it is a

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natural phenomenon as a means of survival strategy 14. Conventional methods of controlling

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the production of biofilm have been practiced based on bacterial killing by antibiotics and

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anti-fouling agents, but the indiscriminate use of antibiotics in strains with increased

80

resistance may lead to mutations in antibiotic resistant strains. Since the anti-fouling agent is

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a non-selective biotoxin, the use of anti-fouling agent causes serious health related damages

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to the environmental ecosystem. It is crucial to continuously monitor the biofilm that the

83

formation from the microorganism for toxicity detection. V. fischeri is one of the most

84

common biosensor used for the risk assessment in aquatic environment 15, therefore, sensing

10, 11, 12

. The resistance to multiple stresses is significantly increased as



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the level of V. fischeri can be used to determine the presence and accumulation degree of

86

specific pollutants as well as to indicate the biofilm formation based on the inhibition of

87

luminescence produced by the bacteria in the presence of toxic substances.

88

Aptamers are single-stranded oligonucleotides (ssDNA or RNA) that fold into stable

89

structural conformations and pattern motif such as stems, loops, hairpins, triplexes or

90

quadruplexes in three-dimensional (3-D) space

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interaction with a given target ranging from small ions to molecular level makes it pragmatic

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in numerous applications for specific detection of various analytes

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antibodies, aptamers have several advantageous properties such as its stability and high

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resistance to enzymes, good tolerance of temperature or pH and extreme of ionic strength,

95

high affinity and specificity to targets in the nanomolar to picomolar range

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of the aptamers to recuperate their native 3-D structure after heat denaturation keeps the

97

aptamer-target complex more stable

98

chemically available in vitro, so that they can be directly applied to a wide variety of targets

99

in short time. The ease in modification process of aptamer makes it effective and detectable

100

16, 17

. The critical ability of aptamers in

18

. Compared to the

19-22

. The ability

23-25

. The synthesis procedure of aptamer is simple and

in many different applications.

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In this study, we demonstrate high sensitivity of the aptamer-based paper strip sensor.

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Paper is easily accessible and can be altered by a wide range of bio-recognition elements

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that include proteins, antibodies, nucleic acids, and various amplification systems. Paper-

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based sensors can be applied across a range of application areas such as in health diagnostics

105

26-29

, environmental monitoring 30, 31, and food quality control 32, 33. The paper strip sensor is

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particularly attractive due to their potential for on-site testing with ease of use, rapidity,

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portability, and elimination of the need for complex detection instrument

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system comprising V. fischeri as an analyte and a pair of DNA aptamer probes was used to

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demonstrate the proof-of-concept on the conventional paper lateral flow strip. Cell-SELEX


34, 35

. A model

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process was conducted to obtain ssDNA aptamers with high specificity and affinity for V.

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fischeri. Aptamers were generated using whole live V. fischeri cells, while Escherichia

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coli and B. subtilis cells were used as negative controls during SELEX processes. After 9

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rounds of selection, the V. fischeri Cell Aptamer (VFCA) pair, VFCA-02 and VFCA-03

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were selected and designed as the detection and capture probe in our aptamer-based paper

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strip sensor. The aptamer-based paper strip sensor facilitates reliable detection of V. fischeri

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and allows rapid and sensitive discrimination between V. fischeri and other bacterial species.

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Results and Discussion

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Cell-SELEX against V. fischeri

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Cell-SELEX procedure was conducted to isolate DNA aptamers from a random

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sequence library to demonstrate its functional binding property to V. fischeri. The aptamers

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were generated by introducing the live V. fischeri cells with a random library, about ~1016

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diverse sequences. The concentration of bound DNA species increased during each selection

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round to further enhance selection towards a high-specificity and high-affinity aptamer pool.

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The nanodrop spectrophotometer analysis was conducted after 9 rounds of SELEX to

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observe and assess the binding affinity of the aptamer pool to V. fischeri. As shown in

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Figure 1, the enrichment pattern was observed during the SELEX processes. Since each

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SELEX round amplifies the aptamer candidates eluted from a previous round, increased

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concentration of eluted DNA aptamers was symbolic of successive SELEX rounds, hence

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enriching the binding affinities progressively. Additionally, negative SELEX step was

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successfully conducted to trim the number of aptamers interacting with non-specific bacteria

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cells and enhance the binding specificity to V. fischeri cells. The 8th selection round was the

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optimal state to isolate aptamer candidates, and no further SELEX rounds were conducted

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after round 9. The efficient exclusion of the non-specific bound aptamer by participating



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count-partners, gram-positive B. subtilis and gram-negative E. coli was a key element of the

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cell-SELEX strategy for V. fischeri. This concept may be applied as a standard procedure for

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specific aptamer selection in whole bacterial cell-SELEX experiment.

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Figure 1. Concentration of eluted V. fischeri DNA aptamers during the cell-SELEX process.

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The

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spectrophotometer. Negative round (N) was performed between round 6 and 7 to prevent

142 143

enrichment of nonspecific binding aptamer for V. fischeri.

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Isolation of functional aptamers against V. fischeri

ssDNA

concentration

of

each

rounds

were

determined

using

Nano-drop

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Sequence alignment was performed to identify 6 groups containing 13 aptamer

146

candidates. To further confirm the binding property of the aptamer candidates against V.

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fisheri, a post-SELEX experiment was conducted. A total 13 aptamer candidates were

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produced, 700 pmoles of ssDNA aptamer dissolved in 200 µL of binding buffer and

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incubated with 108 V. fischeri cells under identical condition. Of the 13 aptamer candidates

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tested, VFAC-02 and VFAC-03 reacted to V. fischeri with high binding ability, as shown in

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Figure 2, and were selected for further investigation. The complete sequences of the 13

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isolated aptamer are provided in Table 1. The secondary structures of the VFCA-02 and


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VFCA-03 aptamers predicted by Mfold show identical hairpin loops in the 5’ region (4-20

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nucleotides) that may play an important role in the binding properties of these aptamers

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(Figure 3).36, 37

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Figure 2. Selection of the optical V. fischeri binding aptamer among the aptamer candidates.

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Among the 13 V. fischeri aptamer candidates of the different sequences with each other, the

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best efficiency of aptamer was chosen using post-SELEX process. The ssDNA of the 13

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aptamer candidates were incubated and eluted with V. fischeri cells. The concentration of the

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eluted 13 aptamer candidates were measured by Nano-drop spectrophotometer. The results

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were determined in triplicates.

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Table 1. List of sequences of the isolated aptamer candidates for Vibrio fischeri. Group

Name

Aptamer sequence (N40)

Size (bp)

VFCA-01

GGTCGTGTGGACTTGCGATTTCGGTTTGGTGTGGTTGGTG

40

VFCA-02

GGGCATGTGGACTTGCGATTTCGGTTTGGTGTGGTTGGGG

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VFCA-03

GGGCATGTGGGCTTGCGATTTCGGTTTGGTGTGGTTGGGG

40

VFCA-04

TGGCATGTGGACTTGCGATTTCGGTTTGGTGTGGTTGGGG

40

VFCA-05

GGGCATGTGGACTTGCGGTTTCGGTTTGGTGTGGTTGGGG

40

VFCA-06

GGGCGTGTGGACTTGCGATTTCGGTTTGGTGTGGTTGGGG

40

VFCA-07

GGGCATGTGGACTTGCGATTTCGGTTGGTGTGGTTGGGG

39

VFCA-08

GGGCATGTGGACTTGCGACTTCGGTTTGGTGTGGTTGGGG

40

Group-02

VFCA-09

AGACAGCATAGCACTGTAACGATTGGTTTGGTGTGGTTGG

40

Group-03

VFCA-10

CCAGAATTGGTGGGTCGACTGCTGGTGTCCTATAAAGGGG

40

Group-04

VFCA-11

CGTGGGCGGTAGAGCCAATGCTACTGGAGCGCGTATCCTA

40

Group-05

VFCA-12

GTCGGAGAGCCCGGCCGTCTGCTCGTAAGTACTATCATAGC

41

Group-06

VFCA-13

GCGGACGGATAGTAAATAGCCCATGTACGCTGCTGGCGTC

40

Group-01

172 173

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Figure 3. Secondary structure prediction (Mfold software) for the highest efficiency aptamers,

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VFCA-02 and VFCA-03. ACS Paragon Plus Environment 8

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Binding of VFCA-02 and VFCA-03 aptamers measured by surface plasmon resonance

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(SPR)

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The binding affinity of the selected aptamers (VFCA-02 and VFCA-03) towards V.

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fischeri was evaluated by surface plasmon resonance (SPR) after immobilization of the

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aptamers on the manufacturer’s SA chip (Figure 4a). Exposure to V. fischeri gave rise to the

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sensorgrams shown in Figure 4b and 4c, resulting in apparent KD values of 1.28 × 10-10 M

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and 1.25 × 10-9 M, respectively. Given the likelihood of multivalent interactions of the cells

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with immobilized aptamers, distinct binding constants cannot be assigned from these data.

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However, assuming equivalent densities of surface attachment, VFCA-02 appears to have a

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significantly greater affinity for V. fischeri compared to VFCA-03.


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Figure 4. Surface Plasmon Resonance experiment was performed for the measurement of binding affinity between V. fischeri and specific

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binding aptamers. (a) Scheme diagram of SA sensor chip immobilized with 5’-biotin modified aptamer. Binding sensorgram of VFCA-02 (b)

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and VFCA-03 (c) against V. fischeri.

10


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To further establish the binding specificity of selected aptamers, an SPR experiment

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was conducted against various live bacterial cells, and their specificity was determined using

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BIA evaluation software. Eight bacterial species Vibrio fischeri, Vibrio parahaemolyticus,

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Escherichia coli, B. subtilis, Shigella sonnei, Staphylococcus aureus, Salmonella

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choleraesuis and Listeria monocytogenes were chosen as a test bacteria panel. The 8 bacteria

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species were grown in appropriate culture media and then harvested in the exponential cell

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growth state. As shown in Figure 5, the interaction of the selected aptamers with these 8

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different bacterial cells was verified. The binding pattern of two aptamers (VFCA-02 and

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VFCA-03) was comparable. Both aptamers had high binding affinity for V. fischeri as

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compared to the binding affinity with other bacterial species. The selected aptamers were

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able to distinguish between pathogenic (V.parahaemolyticus) and non-pathogenic (V.

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fischeri) species.


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Figure 5. Binding specificity of VFCA-02 (a) and VFCA-03 (b) against bacterial species Vibrio fischeri, Vibrio parahaemolyticus, Escherichia

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coli, Bacilllus subtilis, Shigella sonnei, Staphylococcus aureus, Salmonella choleraesuis and Listeria monocytogenes.

12


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Sensitive detection of V. fischeri on aptamer based paper strip sensor

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Figure 5 illustrates the composition and measuring principle of the aptamer based paper

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strip sensor. The paper strip sensor consists of 4 components: 1) sample pad, 2) conjugation

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pad, 3) nitrocellulose membrane pad, and 4) absorbent pad (Figure 6a). A model target V.

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fischeri and two VFCA aptamers (VFCA-02 and VFCA-03), that bind to V. fischeri with

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high specificity were built to exhibit the proof-of-concept. The test solution containing V.

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fischeri cell in SSC buffer was introduced onto the sample pad. The capillary action migrate

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the samples and rehydrated the gold nanoparticles (GNPs)-VFCA-02 aptamer conjugates.

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Then the binding between the GNPs-VFCA-02 aptamer and V. fischeri occurred, resulting in

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formation of V. fischeri-VPCA-02 aptamer-GNPs complexes, and it continued to move

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along the nitrocellulose paper strip. As the complex reached the test line, it was captured by

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the VFCA-03 aptamer immobilized on the test line by interaction between the VFCA-03

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aptamer and V. fischeri. The accumulation of GNPs on the test line results in clearly

225

observed typical red bands (Figure 6b). Once the solution passed through the control line,

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the excess GNPs-VFCA-02 aptamer conjugates were captured on the control line via

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binding events between the streptavidin and the modified biotin in VFCA-02 aptamer.

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Qualitative analysis is conducted by observing the change in color of the test line (Figure

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6b), and quantitative analysis is performed by measuring the color intensities of the red

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bands with a portable paper strip reader. Most of the current paper strip sensors are based on

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the use of antibodies as recognition elements

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strip sensor has several limitations such as low stability on colloical gold particles, low

233

specificity, low conjugation efficiency, and high batch-to-batch variation

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recognition elements have attracted much attention because they are stable over various test

235

conditions and can be chemically synthesized and easily modified to have active chemical

236

groups. Both VFCA-02 and VFCA-03 have been successfully applied to improve the

38

. However, the use of antibodies in paper


39

. Aptamers as


237

binding performance in sandwich format (Figure SI 1).

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To determine the quantitative detection of V. fischeri using the strip sensor, the

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intensities of the test line were estimated and plotted as a function of different concentrations

240

of V. fischeri. Figure 7a represents the photo images of the aptamer based paper strip sensor

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prototype. Different concentrations of V. fischeri were tested to evaluate the sensitivity of

242

the V. fischeri by bioactive paper strip sensor detection method. It was observed that band

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areas increased with increase in the V. fischeri concentration (Figure 7b). In addition, both

244

negative selection cells (B.subtilis and E.coli) didn’t react to V. fischeri aptamer based

245

sandwich formed paper strip sensor (Figure SI 2). The useful analytical range expended

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from 4 × 101 to 4 × 105 CFU/mL of live bacterial cell and was suitable for quantitative work.

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The cartogram in Figure 7c is consistent with the gel electrophoresis and paper strip sensor

248

results. The intensity of the each strip is represented by the peaks in the figure. There is a

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linear relationship between peak height and area with concentration of target cells (R2 =

250

0.9809). This was a significant improvement over the same assay performance, that yielded

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a detection limit of 103~104 CFU/mL

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capability of V. fischeri aptamer based sandwich formed paper strip sensor.

40, 41, 42

. There results demonstrate the extraordinary


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Figure 6. Schematic diagram of the principle for detection of aptamer-based paper strip sensor. (a) GNP-VFCA-02 aptamer-biotin is immobilized on conjugate pad; VFCA-03 aptamer is immobilized on nitrocellulose membrane (test line); Streptavidin is immobilized on

256

nitrocellulose membrane (control line), (b) V. fischeri cell is applied on sample pad with running buffer; (GNP-VFCA-02-biotin + V. fischeri)

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capture of recognition complexes in test line and excess GNPs in control line, results in two red lines.

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15



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Figure 7. (a) Comparison of aptamer-based lateral flow paper and commercial paper strip

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sensor for V. fischeri detection. (b) V. fischeri cells with running buffer were tested using

261 262

aptamer-based paper strip sensor, and test lines were identified with the naked eye. The cell broth added with running buffer at 4×100, 4×101, 4×102, 4×103, 4×104 and 4×105

263

CFU/mL were tested using strip. (c) Linear response to the series of V. fischeri (CFU/mL).

264

The x-axis represents the logarithmic concentration. The y-axis represents the fluorescence

265 266

density. The linear regression correlation coefficient (R2) is 0.9809.

267 268

Experimental section

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Bacteria strains and culture

270

Vibrio fischeri (ATCC 49387), Vibrio parahaemolyticus (ATCC 17802), Bacillus

271

subtilis (ATCC 6051), Escherichia coli (ATCC 29425), Shigella sonnei (ATCC 25931),

272

Staphylococcus aureus (ATCC 25923), Salmonella choleraesuis (ATCC 10708) and Listeria

273

monocytogenes (ATCC 19115) were obtained American Type Culture Collection (ATCC)


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and used in this study. For conventional bacteria culturing, V. fischeri and V.

275

parahaemolyticus were grown at 37°C with containing nutrient broth (BD Difco, UK)

276

supplemented with 3% NaCl. The media for bacteria were cultured as follows; B. subtilis, E.

277

coli, S. sonnei, S. aureus and S. choleraesuis for nutrient broth (BD Difco, UK);

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monocytogene for Brain Heart Infusion (BD Difco, UK). The culture was agitated at 37°C

279

on a shaker at 180 rpm.

L.

280 281

ssDNA Random library: Cell-SELEX

282

The Cell-SELEX process is generally the same as known. A 76-nt combinational DNA

283

aptamer library was obtained from Bioneer (South Korea, Daejeon). The library sequence,

284

location of random (N40) and constant were chemically synthesized: 5′-ATA CCA GCT

285

TAT TCA ATT -N40- AGA TAG TAA GTG CAA TCT-3′, forward primer (5′-ATA CCA

286

GCT TAT TCA ATT) and biotinylated reverse primer (5′-biotin- GA TTG CAC TTA CTA

287

TCT-3’). To label with biotin, the aptamer library was amplified in PCR reactions

288

containing 1× Ex Taq buffer, 0.2 mM dNTP mixture, 5 U Ex Taq DNA Polymerase

289

(TaKaRa, Japan), 10 pM forward primer and 10 pM biotinylated reverse primer in 50 µL of

290

reaction volume. PCR product was purified QIAquick PCR purification kit (Qiagen, USA).

291

To separate from double-strand DNA to single-strand DNA, after 85°C boiling, streptavidin

292

agarose resin (Pierce, USA) was introduced. The result was conducted using 10%

293

polyacrylamide gel electrophoresis containing 0.5 X TBE buffer (data not shown).

294

About 700 pmoles of ssDNA aptamer library was dissolved in 200 µL of binding buffer (1×

295

nutrient broth with 3% NaCl), denatured by boiling at 85°C for 5 minutes and renatured by

296

cooling on RT for 1 hour to form a stable aptamer structure. The aptamer pool was

297

incubated with 108 of V. fischeri cells suspended in 200 µL binding buffer for 1 hour at 4°C

298

with gentle shaking. Aptamer-bound cells were recovered by centrifugation at 13,000 rpm



299

for 10 minutes and washed three times using 500 µL washing buffer (TBS buffer; 10 mM

300

Tris-HCl, 0.85% NaCl, pH 8.0) to remove unbound and weakly bound aptamers. To obtain

301

V. fischeri cell-bound aptamer, the cell-aptamer was resuspended in elution buffer (TE

302

buffer; 10 mM Tris-HCl, 1 mM EDTA, pH 8.0), boiled at 85 °C for 10 minutes, cooled on

303

ice, and centrifuged. The eluted ssDNA in supernatant was recovered by phenol/chloroform

304

extraction and ethanol precipitation and then used as the template DNA for the next round of

305

selection.

306

To assure the specificity of aptamer, negative-SELEX was conducted after the 6th round of

307

Cell-SELEX process. The aptamer pool (700 pmols) suspended in 200 µL binding buffer

308

was incubated with nonspecific cell mixture (108 of B. subtilis and 108 of E. coli) for 1 hour

309

at 4°C with gentle shaking. The aptamer-bound cells were discarded, while the unbound

310

aptamers in supernatant were collected for next round.

311 312

Identification of V. fischeri binding aptamers

313

Following 9 rounds of Cell-SELEX and one of the negative-SELEX, the obtained

314

aptamer pool was measured the eluted ssDNA concentrations from each round using Nano-

315

drop spectrophotometer (Thermo scientific, USA). The eluted aptamer pool of the each

316

round were quantified individually on the three times. The purified aptamer pool of optimal

317

round (8th round) was ligated into the T-blunt vector using T-blunt cloning kit (Solgent,

318

South Korea). The ligated vectors were transformed into E. coli DH5α (Solgent, South

319

Korea) and cells were incubated on Luria-Bertani (LB) agar plates with ampicillin (50 µg/ml)

320

and kanamycin (50 µg/ml) at 37°C for 15-20 hours. Individual colonies of the transformed

321

cells were cultured 5 ml LB broth with ampicillin (50 µg/ml) and harvested for extraction of

322

plasmid DNA using DNA-spin plasmid DNA purification kit (Invitrogen). Sequencing of

323

the plasmid DNA of the selected transformants was performed by Bioneer Inc., Daejeon,


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43

324

Korea. The sequences were analyzed by ClustalX sequence alignment program

. The

325

identified aptamers of individual sequence were prepared for the Post-SELEX 20. A total 13

326

aptamer candidates were produced 700 pmols of ssDNA aptamer dissolved in 200 µL of

327

binding buffer and incubated with 108 V. fischeri cells under identical condition. After

328

washing, each aptamer-cells complex was prepared for PCR amplification. The

329

amplification condition for PCR was as follows: an initial denaturation at 95ºC for 1 min

330

followed by 20 amplification cycles of 45 s of denaturation at 94ºC, 1 min annealing at 55ºC,

331

1 min elongation at 72ºC and final elongation of 5 min at 72ºC. The eluted aptamer

332

concentration was measured to obtain the best efficiency aptamer.

333 334

Secondary structure prediction

335

To further confirm the binding affinity of the aptamer candidates against V. fischeri,

336

post-SELEX step was conducted. The most efficient selection of two aptamers (VFCA-02,

337

VFCA-03) were calculated the secondary structure. The secondary structures of the single-

338

stranded DNA aptamers were predicted using Mfold (http://unafold.rna.albany.edu/) by free

339

energy minimization algorithm.

340 341

Aptamer binding affinity analysis by Surface Plasmon Resonance

342

The streptavidin immobilized sensor chip SA (GE Healthcare for Biacore 3000) was

343

used for the interaction test. The 5’-biotin-modified DNA aptamers for V. fischeri (VFCA-

344

02 and VFCA-03) were immobilized by passing 500 nM aptamers for 10 minutes at 10 µL

345

per min on SA chip. 1 mL of HBS-EP buffer (GE Healthcare, UK) was used for pre-

346

activation of the SA chip, at a flow rate of 10 µL per min for 7 minutes. The SA chip was

347

also pre-activated at a flow rate of 10 µL per min for 10 minutes with 1 mL of the running

348

buffer. Then, the 5′-biotinylated aptamers were immobilized on the chip as recommended by



349

manufacturer’s manual. To observe the consecutive interaction using the BIACORE

350

instrument, all procedures were automatically implemented to create repetitive cycles of

351

sample injection (90 µL injection samples, at a flow rate of 10 µL per minutes) and

352

regeneration (1 M NaCl, 50 mM NaOH, 50% Isopropanol), according to the instruction

353

guidelines (BIA evaluation program, version 4.1).

354 355

Aptamer-based paper strip sensor

356

Preparation of Gold Nanoparticles (GNPs)-VFCA aptamer conjugates: The preparation

357

of GNPs-aptamer conjugations was based on the formation of carboxyl-amine bond. The

358

modification of 5’ aminated and 3’ biotinylated VFCA-02 aptamer was used for conjugation

359

with 10 ± 2-nm-diameter carboxylic acid functionalized GNPs (Sigma, USA). 500 µL of

360

COOH-PEG-GNPs solution with OD 0.3 were mixed with 50 mM EDC/NHS solutions (GE

361

healthcare, UK) for GNPs activation. The reaction was left to proceed at 25°C for 60

362

minutes; it was then diluted and centrifuged at 10,000 rpm for 15 minutes. A 1 mL portion

363

of same concentration of modified VFCA-02 aptamer solution (10 µM) was added in to the

364

activated GNP solution and reacted at 25°C for 16 hours. Then, a 2 M NaCl solution was

365

added to a final concentration of 50 mM and reacted for 12 hours. The mixture was

366

centrifuged by 6,500 rpm for 15 minutes. The GNP-VFCA-02 aptamer conjugation solution

367

was stored at 4°C for further study.

368

Fabrication of the VFCA aptamer-based strip: The biosensor strip for detecting V.

369

fischeri consists of the following components: sample pad, conjugation pad, absorption pad,

370

and nitrocellulose membrane. The sample pad (3 mm × 13.5 mm) was contrived from

371

cellulose fiber (EMD Millipore, USA) and soaked with the 1× SSC buffer containing 0.25%

372

Triton X-100, 50 mM Tris-HCl, 1% Tween 20 and 150 mM NaCl. It was then dried and

373

stored in desiccators at room temperature. The conjugate pad was prepared by dispensing a


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374

desired volume of GNP-VPCA-02 aptamer conjugate solution onto the conjugate pad. The

375

test line and control line on the nitrocellulose membrane (3 mm × 15 mm) were prepared by

376

dispensing the VFCA-03 aptamer (test line) and streptavidin (control line) solutions. The

377

distance between two lines was approximately 2.5 mm. The membrane was dried at room

378

temperature for 1 hour and stored at 4°C.

379

Assay procedure and detection of V. fischeri: The detection of V. fischeri was conducted

380

by applying 100 µL appropriate standard test V. fischeri cell culture broth to the strip. For

381

each specimen, V. fischeri culture (100 µL of 10-fold dilutions in 1× SSC buffer to range

382

from 4 × 105 to 4 × 100 CFU/mL) were added onto the sample pad, and the solution

383

migrated towards the absorption pad. The test line and control line were evaluated visually

384

within 10 min. For quantitative measurements, the optical intensity of the red bands was

385

read using the detector instrument “I-CHROMA” (Point of Care reader RF203 was

386

purchased from Boditech Med incorporated) The assays were conducted in triplicate using

387

the test strip. The test and control line turning red is recorded as positive sample, indicating

388

the presence of V. fischeri. If the control line but not the test line is colored, the sample is

389

considered negative. If only the red test line appears, the strip is considered invalid, and the

390

test should be repeated using new strip.

391 392

Conclusion

393

We have demonstrated an aptamer-based paper strip sensor using V. fischeri as a marine

394

environmental bioindicator. The specific aptamers, VFCA-02 and VFCA-03 were selected

395

for detecting V. fischeri by using the cell-SELEX process. We used a post-SELEX procedure

396

for the highly efficient selection of these aptamers. The paper-strip sensor shows a significant

397

ability to detect and discriminate V. fischeri from other bacterial species. The useful

398

analytical range of the sensor expended from 4 × 101 to 4 × 105 CFU/mL of target cell,



399

suitable for quantitative work. The results demonstrate the extraordinary selective and

400

sensitive detection capability of V. fischeri aptamer based sandwich formed paper strip sensor.

401 402

Supporting Information

403

Fluorescent intensity of aptamer-based sandwich assay with various controls, Experimental

404

details for fluorescent assay and Aptamer-based paper strip test for negative selection cells.

405 406

Acknowledgements

407

This study was performed by the support of the "Cooperative Research Program for

408

Agriculture Science & Technology Development (Project No. PJ012568)” (Rural

409

Development Administration, Republic of Korea). This work was also supported by the

410

National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT

411

& Future Planning (NRF-2015R1A4A1041869). The authors are grateful for their support.

412 413

Author Contributions

414

J-Y.A., J. M., and Y-H.K. conceived and designed the experiments; W-R.S. and Y-H.K.

415

conducted the experiments; W-R.S., S.S.S., and J-H.K. analyzed the data; S-K.R., J-Y.A.,

416

and J. M. contributed reagent/analysis tools; J. M., J-Y.A., and Y-H.K. wrote the study.

417 418

Conflicts of Interest: The authors declare no conflict of interest.

419 420

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