Aptamer-Based Paper Strip Sensor for Detecting Vibrio fischeri - ACS

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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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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

2

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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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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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

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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

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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

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

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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

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

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

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

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

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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,

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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

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aptamer-target complex more stable

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

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

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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

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, 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

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. 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

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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

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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

40

VFCA-03

GGGCATGTGGGCTTGCGATTTCGGTTTGGTGTGGTTGGGG

40

VFCA-04

TGGCATGTGGACTTGCGATTTCGGTTTGGTGTGGTTGGGG

40

VFCA-05

GGGCATGTGGACTTGCGGTTTCGGTTTGGTGTGGTTGGGG

40

VFCA-06

GGGCGTGTGGACTTGCGATTTCGGTTTGGTGTGGTTGGGG

40

VFCA-07

GGGCATGTGGACTTGCGATTTCGGTTGGTGTGGTTGGGG

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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

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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.

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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.

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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

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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

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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

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

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

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. However, the use of antibodies in paper

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. Aptamers as

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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

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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

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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

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

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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

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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 =

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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.

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. 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

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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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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

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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

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

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

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

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Experimental section

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Bacteria strains and culture

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

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

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

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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.

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

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

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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

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on a shaker at 180 rpm.

L.

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ssDNA Random library: Cell-SELEX

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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selection.

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

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

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

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

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aptamers in supernatant were collected for next round.

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Identification of V. fischeri binding aptamers

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

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

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

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

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

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

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

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

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

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

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

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Korea. The sequences were analyzed by ClustalX sequence alignment program

. The

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

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

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

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

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

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

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

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concentration was measured to obtain the best efficiency aptamer.

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Secondary structure prediction

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

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

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

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

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energy minimization algorithm.

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Aptamer binding affinity analysis by Surface Plasmon Resonance

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

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

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

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

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

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

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

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

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

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

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

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guidelines (BIA evaluation program, version 4.1).

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Aptamer-based paper strip sensor

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

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

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

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

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

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

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

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

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

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

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

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was stored at 4°C for further study.

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

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

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

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

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

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

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

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

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

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

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temperature for 1 hour and stored at 4°C.

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

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

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

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

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

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

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

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

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

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

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

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test should be repeated using new strip.

391 392

Conclusion

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

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

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

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

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

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

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

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

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Supporting Information

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

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

405 406

Acknowledgements

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

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

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

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

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

412 413

Author Contributions

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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.,

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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.

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