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Patterned Photonic Nitrocellulose for Pseudo-Paper ELISA Junjie Chi, Bingbing Gao, Mi Sun, Fengling Zhang, Enben Su, Hong Liu, and Zhongze Gu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01732 • Publication Date (Web): 16 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017

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Analytical Chemistry

[Prepared for publication as an Article in Analytical Chemistry]

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Ms. ID:

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Patterned Photonic Nitrocellulose for Pseudo-Paper ELISA

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Junjie Chi, † Bingbing Gao, † Mi Sun, † Fengling Zhang, † Enben Su#,

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Hong Liu,*,†,‡ and Zhongze Gu*,†,‡

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† State Key Laboratory of Bioelectronics, School of Biological

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Science and Medical Engineering, Southeast University, Nanjing

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210096, China

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‡ Laboratory of Environment and Biosafety, Research Institute of

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Southeast University in Suzhou, Suzhou 215123, China

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# Getein Biotech, Inc. No.9 Bofu Road, Luhe Distric, Nanjing,

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Jiangsu, China (211505)

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*To whom correspondence should be addressed.

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Email: [email protected], [email protected]

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Submitted: June 15, 2017

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Abstract

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We report an enzyme-link immunosorbent assay (ELISA) based on

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patterned pseudo-paper that is made of photonic nitrocellulose

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for highly sensitive fluorescent bioanalysis. The pseudo-paper is

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fabricated using self-assembled monodisperse SiO2 nanoparticles

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that are patterned on a polypropylene substrate as template. The

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self-assembled nanoparticles have a close-packed hexagonal (opal)

8

structure, so the resulting nitrocellulose has a complementary

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(inverse opal) photonic structure. Owing to the slow-photon

10

effect of the photonic structure, fluorescent emission for ELISA

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is enhanced by up to 57-fold without increasing the assay time or

12

complexity. As the detection signal is significantly amplified, a

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simple smartphone camera suffices to serve as the detector for

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rapid and on-site analysis. As a demonstration, human IgG is

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quantitatively analysed with a detection limit of 3.8 fg/mL that

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is lower than that of conventional ELISA and paper-based ELISA.

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The consumption of sample and reagent is also reduced by 33-times

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compared with conventional ELISA. Therefore, the pseudo-paper

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ELISA based on patterned photonic nitrocellulose is promising for

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sensitive, high-throughput bioanalysis.

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1

Introduction

2

Enzyme-link immunosorbent assay (ELISA) was introduced by

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Perlmann and Engvall in 1971. In a typical sandwich ELISA, a

4

solid substrate is functionalized with capture antibody, which

5

binds to its target antigen in an aqueous sample. An additional

6

enzyme-linked tracing antibody is then added to bind to the

7

antigen forming a sandwich structure, and the enzyme amplifies

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the detection signal by a catalytic reaction. Combining the high

9

specificity of the antibody-antigen binding and the high-turnover

1

2

10

catalytic reaction of the enzyme, ELISA has been proven to be a

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powerful bioanalytical tool for a wide range of biochemical

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applications.

13

2, 3

Conventional ELISA typically involves enzyme substrates that 4,

14

produce observable signals to indicate the presence of analyte.

15

5

16

plate fabricated by injection molding of polystyrene (PS). The

17

whole assay process can be fully automated to accomplish high-

18

throughput testing. However, it usually requires large amount of

19

sample and reagents (~ 20-200 µL).6 The heterogeneous assay also

20

involves long-time reaction steps (~ 1 h) because reagents in the

21

bulk solution have to diffuse over a long distance to react with

22

the immobilized substance on the surface.6 The detection limit is

23

still not low enough for some important diagnostic biomarkers

24

even using sophisticated and expensive instruments,7-11 although

25

various detection methods have been developed to improve the

26

sensitivity, such as chemiluminescent,12 electrochemical,13 Raman,14

27

plasmonic,15 and fluorescent methods.16

The whole ELISA is usually carried out in a standard 96-well



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Other than plastic materials, cellulose-based materials have

2

also been used for immunoassays for a long time. For example,

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dot-immunobinding assays using filter paper and lateral-flow

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immunochromatographic assays using nitrocellulose paper have been

5

available for decades.

6

renewable and biodegradable cellulose-based materials are

7

relatively more environmental friendly, and they provide more

8

biocompatible surface for maintaining the activity of biological

9

reagents such as antibodies and enzymes.

17, 18

Compared with plastic materials, the

19

Recently, there has

10

been considerable interest in using patterned paper as the

11

substrate for ELISA owing to several significant advantages.20,

12

The fabrication of patterned paper that mimics the standard 96-

13

well plate can be accomplished simply by printing which is

14

scaleable and may enable cost-effective production. The ELISA

15

based on patterned paper requires smaller amount of reagents and

16

sample owing to the small volume of the paper reservoir. The

17

reduced volume leads to short distance that reagents have to

18

diffuse to react with immobilized substance, so the assay time

19

can be remarkably shortened.22 However, compared with conventional

20

PS-based ELISA, the most crucial drawback of paper ELISA is the

21

low sensitivity owing to small volume of the paper reservoir.

22

Usually, the sensitivity of paper-based ELISA is lower than

23

conventional ELISA by approximately an order of magnitude.22 What

24

makes it worse is that the reproducibility of paper-based ELISA

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is usually worse than PS-based ELISA leading to higher detection

26

limit. Therefore, development of paper-based ELISA with high

27

detection sensitivity is highly of importance. 4 ACS Paragon Plus Environment

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We have previously reported the fabrication of patterned

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photonic nitrocellulose for pseudo-paper microfluidics. The

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photonic structure of the patterned nitrocellulose resulted in

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several highly-wanted characteristics for microfluidic

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bioanalysis.

6

nitrocellulose for high-throughput pseudo-paper ELISA (Figure 1).

7

The fabrication of the patterned photonic nitrocellulose is based

8

on self-assembly techniques. What differentiates the

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nitrocellulose pseudo-paper from conventional nitrocellulose

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Here, we report the use of the patterned photonic

10

paper is that the interconnected pores of the pseudo-paper are

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highly ordered. The periodic structure of the material magnifies

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the interaction of light with fluorophores leading to amplified

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fluorescent emission.

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enhancement effect of the photonic nitrocellulose, and its

15

application to highly sensitive fluorescent ELISA of a biomarker.

We investigated the fluorescent-

16 17

Experimental Section

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Chemicals and materials. Nitrocellulose was obtained from

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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). TWEEN-20,

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acetone, dimethylformamid (DMF), ethylene alcohol, bovine serum

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albumin (BSA), horseradish peroxidase (HRP), H2O2 and HF were

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purchased from Sigma-Aldrich. Polypropylene (PP) film (PP2910,

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100 µm thick, 210 mm × 297 mm) was obtained from 3M Company (St.

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Paul, MN). Monodisperse SiO2 nanoparticles with a diameter of

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247, 302, 323, and 340 nm respectively were synthesized using the

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Stöber-Fink-Bohn method.24 Rabbit anti-human IgG, human IgG, HRP-

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labelled mouse anti-human IgG and FITC-labelled goat anti-human 5 ACS Paragon Plus Environment


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IgG were all purchased from Santa Cruz Biotechnology Co., Ltd

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(Shanghai, China). 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) was

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purchased from AAT Bioquest, Inc (Sunnyvale, US). 0.10 M PBS (pH

4

7.4) containing 0.20% TWEEN-20 (PBST) was used for wash. All

5

solutions were prepared with deionised water (18.0 M• cm, Milli-Q

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Gradient System, Millipore) with ultraviolet sterilization. All

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reagents were used as received without further purification.

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Experimental Procedures. The fabrication of the patterned photonic nitrocellulose was based on the procedure we have 23

10

previously reported with some modification.

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treated in a plasma cleaner (DT-01, Opsplasma, China) to increase

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its surface hydrophilicity (5 min, 100 w). Next, a toner pattern

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with the format of standard 96-well plate was printed on the PP

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substrate using an office laser printer (CP105b, Fuji Xerox). A

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0.50 µL aliquot of aqueous solution containing 20% (w/v)

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monodisperse SiO2 nanoparticles, 13% (v/v) ethylene alcohol and

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0.050% TWEEN-20 was dropcast onto each well.

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hydrophobicity of toner printed on the substrate, the solution

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was restricted in the hydrophilic well surrounded by the printed

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toner. After complete drying at a room temperature of 25 oC, the

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colloid crystal of SiO2 nanoparticles formed in the well.

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To obtain the photonic nitrocellulose which has the

First, PP film was

Due to the

23

structures complementary to the colloid crystal, an acetone and

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DMF (1:1 v/v) solution containing 7.5% (w/v) nitrocellulose was

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dropcast onto the PP film to form a layer of nitrocellulose on

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the PP film. After baking at 60 oC for 6 h, the nitrocellulose

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layer containing SiO2 nanoparticles was torn off from the PP 6 ACS Paragon Plus Environment

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film. Then the SiO2 nanoparticles remaining on the nitrocellulose

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layer were etched using an aqueous solution containing 4.0% HF.

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Finally, the nitrocellulose membrane was thoroughly washed with

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deionised water and then dried to obtain the photonic

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nitrocellulose for pseudo-paper ELISA. For control experiment,

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the nitrocellulose solution was directly dropcast onto the PP

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substrate without the SiO2 colloid crystal template resulting in a

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random-structured nitrocellulose membrane. The reflection spectra

9

of these materials were obtained using an optical-fibre

10

spectrometer (QE 65000, Ocean Optics). Scanning electron

11

microscopy (SEM, Hitachi S-3000N, Japan) was used to obtain the

12

electronic micrographs of these materials.

13

To prepare the substrate for pseudo-paper ELISA, a 3.0 µL

14

aliquot of 100 µg/mL rabbit anti-human IgG (capture antibody) was

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dropcast onto each photonic nitrocellulose well. After incubating

16

in an enclosed chamber at a room temperature of 25 oC for 30

17

minutes, the substrate was washed for 3 times by dropcast 0.10 M

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PBST solution (pH 7.4) containing 0.20% TWEEN-20 on the reservoir

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with gentle shaking to remove the free antibody. Next, a 3.0 µL

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aliquot of 0.10 M PBS solution (pH 7.4) containing 1.0% BSA was

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dropcast onto each well for blocking the remaining active spots.

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A 3.0 µL aliquot of testing sample containing human IgG was

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dropcast onto each well. After incubation in an enclosed chamber

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at a room temperature of 25 oC for 30 minutes, the substrate was

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washed for 3 times with 0.10 M PBST solution (pH 7.4) containing

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0.20% TWEEN-20. The excess wash buffer was removed using a piece

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of blotting paper. A 3.0 µL aliquot of 20 µg/mL HRP-labelled 7 ACS Paragon Plus Environment


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mouse anti-human IgG was added. The incubation and wash procedure

2

were the same as mentioned above. Finally, a 3.0 µL aliquot of

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100 µM fluorogenic solution containing ADHP and H2O2 was dropcast

4

onto the well. After incubation in a dark chamber at 37 C for 30

5

minutes, the fluorescence intensity was measured using a

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fluorospectro photometer (F-7000, Hitachi). A fluorescent

7

microscope (BX53, Olympus) was used to obtain the fluorescent

8

image of the detection zone.

9

o

A home-made apparatus for smartphone-based fluorescent

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detection was designed using 3D MAX software and fabricated using

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a three-dimensional printer (Maker Bot Replicator) and polylatic

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acid (PLA) as the printing material. The apparatus consisted of a

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green LED (Edmund Optics, USA) as the light source, 530±10 nm

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bandpass filter for the light source and 585 nm longpass filter

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(Giai photonics Co. Ltd., China) for the smartphone camera.

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Results and Discussion

3

Paper is a cellulose-based material that is used as the substrate

4

for a range of chemical analysis such as pH test, iodine-starch

5

test, urine dipstick test, chromatography and lateral-flow assays.

6

In this work, we used nitrocellulose, which is a cellulose

7

derivative, as a starting material to fabricate the substrate for

8

ELISA. We first printed toner on a hydrophilic PP substrate so

9

that an 8×12 array of reservoirs was patterned. Then the photonic

10

nitrocellulose was prepared in each reservoir based on self-

11

assembly technique.

12

The obtained photonic 96-well ELISA substrate was shown in

13

Figure 2a (I). The nitrocellulose formed a green colored layer in

14

the reservoir with only a hole in the central probably owing to

15

coffee-ring effect, as shown in Figure 2a (II). The green color

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of the nitrocellulose reservoir was because of the Bragg

17

scattering effect of the periodic nanostructure of the material

18

leading to a photonic stopband. Since the nitrocellulose layer

19

was more hydrophilic than the surrounding toner layer, aqueous

20

sample was confined in the reservoir without leaking during the

21

ELISA process. After completion of ELISA, the generated

22

fluorescent species can enter the nanopores of the nitrocellulose

23

which caused color change of the photonic crystals (PCs) (Figure

24

2a (III)). By attaching the nitrocellulose substrate to a plastic

25

backing substrate, the fabricated photonic pseudo-paper can

26

replace the conventional 96-well plate to be used in a



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commercially available ELISA processor for automated high-

2

throughput testing (Figure 2b (I and II)).

3

PCs have been widely demonstrated to be an effective and

4

efficient method to amplify fluorescent emission based the slow-

5

photon effect.

6

small near the edge of the photonic stopband. Under the

7

circumstance, photons can couple with a local resonance and Bragg

8

scattering, which greatly enhances the interaction of light with

9

substrate. In addition, the high density of states near the

25

The group velocity of light becomes abnormally

10

photonic stopband enhances the coupling of spontaneously emitted

11

photons to these mode.26,

12

enhanced at the band edge but suppressed inside the gap.

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The stopband of

27

Thus fluorescence can be significantly

photonic material can be tuned over a wide

14

range for different fluorophores by changing the crystal

15

structure, the refractive indices of the material and the

16

dimensions of the structure.28 For the photonic nitrocellulose

17

which has the inverse-opal structure, the stopband is mainly

18

governed by the diameter of the colloidal spheres used to

19

fabricate the structure according to the Bragg equation,

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λ=1.633×dnavg., where d is the center-to-center distance between

21

neighboring particles, and navg. is the average refractive index of

22

the material.29

23

To study the fluorescence enhancement property of the

24

photonic nitrocellulose with different stopbands, we fabricated

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different photonic nitrocellulose reservoirs by changing the

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diameter of the SiO2 nanoparticle template (i.e. 247, 302, 323

27

and 340 nm). Figure 3a showed the SEM images of the SiO2 colloid 10 ACS Paragon Plus Environment

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crystal templates having close-packed hexagonal structures and

2

the corresponding photonic nitrocellulose fabricated using these

3

templates. Note that the nanopores of the top layer were

4

interconnected with those of the underlying layers so that the

5

solutes can diffuse into the photonic nitrocellulose layer. For

6

control experiments, a nitrocellulose membrane fabricated with no

7

template was also fabricated, which had a random porous structure.

8 9

The photonic nitrocellulose reservoirs showed different colors (Figure 3b). The colors (V-VIII) were from the highly

10

ordered structures of the nitrocellulose reservoirs which

11

resulted in photonic band gaps. With the band gap, photons with

12

specific wavelength cannot pass through the material and are

13

reflected, shown in Figure 3c. Half-peak width of the reflection

14

spectra indicates the degree of order of the nanostructure, which

15

had significant influence on the light propagation and

16

interaction with fluorescent molecules.30 Compared with photonic

17

nitrocellulose we fabricated, the random-structured

18

nitrocellulose showed broad reflection from 350 to 700 nm owing

19

to diffuse reflection of the surface.

20

The maximum excitation and emission wavelengths (λex and λem)

21

for the AHOP generated from ADHP oxidation, were 571 nm and 585

22

nm, respectively, which was shown in Figure 3c. To evaluate the

23

effect of the stopband wavelengths on the fluorescent intensity,

24

we dropcast the same amount (3.0 µL) of solution containing 100

25

µM fluorogenic substrate (ADHP), 100 µM H2O2 and 1 ng/mL HRP-

26

labelled mouse anti-human IgG into each pseudo-paper reservoir

27

and allow them to react at 37 oC in a dark place for 30 minutes. 11 ACS Paragon Plus Environment


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Figure 4 showed fluorescence intensities of AHOP on different

2

substrates. The fluorescent intensity obtained from each of the

3

photonic pseudo-paper was obviously higher than that of random-

4

structured nitrocellulose. Furthermore, the photonic

5

nitrocellulose with the stopbands slightly away from the λex and

6

λem (i.e. 435 nm, 545 nm and 600 nm) lead to significant

7

enhancement of fluorescnece. The greatest enhancement (about 57-

8

fold) was observed for photonic nitrocellulose with a stopband of

9

545 nm, which was used for further ELISA experiments. For the

10

photonic nitrocellulose with the stopband (585 nm) overlapped

11

with both the λex and λem, the fluorescent enhancement was

12

suppressed as we have previously discussed.

13

For the fluorescent ELISA, capture antibody for human IgG

14

was immobilized on the photonic nitrocellulose, and the

15

nitrocellulose was then blocked by BSA. The SEM of the

16

nitrocellulose before and after antibody immobilization was shown

17

in Figure 5a. A layer of protein was observed after

18

immobilization of antibody. The adsorbed protein might result in

19

the shift of the stopband of the nitrocellulose because of it

20

changes the refractive index in the nanopores. Therefore, the

21

reflectance spectra of the photonic nitrocellulose before and

22

after antibody immobilization were obtained and shown in Figure

23

5b. There was just several nanometers’ red-shift of the stopband

24

after antibody immobilization, so its’ influence on assay results

25

was negligible.


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1

We carried out an ELISA for the detection of human IgG on

2

the prepared photonic pseudo-paper. As shown in Figure 6, the

3

fluorescent intensity measured from the detection reservoir

4

increased with increasing concentration of human IgG. A linear

5

correlation between the fluorescent intensity and the logarithm

6

of the human IgG concentration was obtained from 1×10-2 to 1×103

7

pg/mL. The limit of detection (LOD), calculated as 3 times the

8

standard deviation of the testing results of the blank divided by

9

the slope of the calibration curve, is 3.8 fg/mL (2.5×10-2 fM),

10

which is significantly lower than commercial ELISA and paper-

11

based ELISA (Table 1). Moreover, owing to the small volume of the

12

detection reservoir, the consumption of sample and reagents was

13

remarkably reduced to 3.0 µL, which may enable lower cost tests.

14

Since the fluorescent intensity was significantly enhanced

15

using the photonic pseudo-paper as the substrate, a smartphone

16

camera suffices to detect the fluorescent signal. A home-made

17

device was designed to carry out the on-site detection. The

18

device was comprised of a LED as the light source and two filters

19

for excitation and emission, respectively. The smartphone was

20

equipped with a normal CMOS camera with 13 megapixel resolution

21

to acquire images (Figure 7a). A smartphone App was programmed to

22

recognize the detection reservoir, and then analyze the RGB value

23

for determination of the concentration of IgG in the sample

24

(Figure 7b). This portable and cost-effective mobile detector

25

could be useful for inexpensive, point-of-care testing under

26

resource-limited conditions.



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1 2

Summary and Conclusions

3

In summary, we have reported the patterned pseudo-paper for

4

carrying out ELISA with high sensitivity and low consumption of

5

sample and reagents. The stopband and fluorescent enhancement

6

properties of the photonic nitrocellulose pseudo-paper with

7

different pore diameters were investigated. Due to the slow-

8

photon effects of the photonic nitrocellulose that enhanced the

9

interaction of the photons with the fluorophore, the fluorescent

10

emission was amplified by up to 57-fold in comparison with

11

random-structured nitrocellulose. Using the material as the ELISA

12

substrate, human IgG was quantitatively detected with a LOD of

13

3.8 fg/mL. A smartphone-based system was also designed for

14

inexpensive, on-site detection. Therefore, the pseudo-paper ELISA

15

is sensitive, fast, cost-effective, and environmental friendly

16

than conventional ELISA based on plastic 96-well plate. We

17

believe it is promising for quantitative bioanalysis.

18 19


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1 2

Acknowledgments

3

We

4

Recruitment

5

Entrepreneurial Talent Recruitment Program of Jiangsu Province,

6

the

7

21327902, 21635001), the Natural Science Foundation of Jiangsu

8

(BK20140619), the Science and Technology Development Program of

9

Suzhou (ZXY201439), the Research Fund for the Doctoral Program of

10

Higher Education of China (20120092130006), State Key Project of

11

Research

12

Special

13

Scientific and Technological Achievements (BA2015067).

gratefully

National

and Funds

acknowledge

Program

Natural

of

Global

Science

Development of

financial

Jiangsu

support

Experts,

Foundation

of

(2016YFF0100802). Province

for

the

14 15


from

Chinese

Innovative

China

The

and

(21405014,

Project

of

Transformation

of


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1 2 3 4

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Wang, X.; Xu, G., Analysis of pirlimycin residues in beef

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muscle, milk, and honey by a biotin–streptavidin-amplified

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1 2 3

Figure Captions Figure 1. Schematic illustration showing the photonic pseudo-

4

paper and fluorescent ELISA carried out on the substrate.

5 6

Figure 2. a) A photograph of the photonic pseudo-paper with the

7

format of a standard 96-well plate fabricated using SiO2 colloid

8

crystal (diameter: 302 nm) as the template (I). Scale bar: 1 cm.

9

A photograph of a detection reservoir before (II) and after (III)

10

completion of the ELISA process. Scale bar: 1 mm. b) A

11

conventional plastic 96-well plate for automated ELISA (I). A

12

photonic pseudo-paper attached on a flat substrate for automated

13

ELISA with fluorescent enhancement (II). Scale bar: 5 cm.

14 15

Figure 3. a) Scanning electron micrographs of the SiO2 template

16

used to fabricate the photonic nitrocellulose (from I to IV), the

17

corresponding as-prepared photonic nitrocellulose (from V to

18

VIII) and the randomly-structured nitrocellulose membrane

19

fabricated by directly dropcasting nitrocellulose solution onto

20

the PP film and allowing it to dry (IX). The SiO2 template were

21

fabricated by self-assembly of monodisperse SiO2 colloid with a

22

diameter of 247 nm, 302 nm, 323 nm and 340 nm, respectively.

23

Scale bar: 500 nm. b) Optical photographs of the photonic

24

nitrocellulose ELISA reservoir. Scale bar: 1 mm. c) Reflectance

25

spectra of the nitrocellulose substrates. The excitation and

26

emission spectra of AHOP are indicated using dashed lines. The



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

reflection peak was 435 nm (V), 545 nm (VI), 585 nm (VII) and 600

2

nm (VIII), respectively.

3 4

Figure 4. a) Fluorescent spectra of the AHOP on the

5

nitrocellulose substrates. b) The maximum fluorescent intensity

6

as a function of the nitrocellulose structures. The error bars

7

represent standard deviation for three replicated measurements.

8 9

Figure 5. a) Scanning electron micrographs of the photonic

10

nitrocellulose fabricated for ELISA before (I) and after (II) the

11

immobilization of capture antibody and BSA blocking. Scale bar:

12

500 nm. b) Reflectance spectra of the nitrocellulose substrate

13

before (VI) and after (VI’) the antibody and BSA modification.

14 15

Figure 6. a) Fluorescence intensity measured after completion of

16

the ELISA as a function of the concentration of human IgG in the

17

sample. b) The fluorescence intensity as a function of the

18

logarithm of the IgG concentration. The error bars represent

19

standard deviation for three replicated measurements. Inset:

20

fluorescent micrographs obtained for the ELISA. Scale bar: 1 mm.

21 22

Figure 7. Smartphone-based device for fluorescent detection. a)

23

photographs of the device. Scale bar: 1 cm. b) The screenshots of

24

the smartphone showing the detection App.

25 26

Table 1.

27

conventaionl cellulose and PS.22,

Comparison between ELISAs using the pseudo-paper, 31


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1


Figures

2 3

Figure 1 / Chi et al.

4



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1

2 3 4


5 6


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1 2 3


4 5 6



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3


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1 2


3



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3


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1 2


3 4 5



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Page 30 of 31

1 Photonic NC-based

Paper-based

Conventional

ELISA

ELISA

ELISA

LOD

2.5×10 fM

54 fM

1.07 pM

Samples and reagents

Volume (µL)

Volume (µL)

Volume (µL)

1)Capture antibody

3.0

3.0

100

2)Blocking

3.0

3.0

100

3)Samples

3.0

3.0

50

4)Second antibody

3.0

3.0

100

5)Enzyme substrates

3.0

3.0

150

Total

15

15

500

-2

immobilization

2 3

Table 1 / Chi et al.

4 5


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1 2

TOC Graphic

3

4 5


Patterned Photonic Nitrocellulose for Pseudopaper ELISA - American

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