Generating Microdroplet Array on Photonic Pseudo-Paper for Absolute

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Functional Nanostructured Materials (including low-D carbon)

Generating Microdroplet Array on Photonic PseudoPaper for Absolute Quantification of Nucleic Acids Junjie Chi, Changmin Shao, Xin Du, Hong Liu, and Zhongze Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11552 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018

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ACS Applied Materials & Interfaces

Generating Microdroplet Array on Photonic PseudoPaper for Absolute Quantification of Nucleic Acids

Junjie Chi, Changmin Shao, Xin Du, Hong Liu*, and Zhongze Gu* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China *E-mail: [email protected] *E-mail: [email protected] KEYWORDS: pseudo-paper, microdroplet array, digital LAMP, absolute quantification, photonic crystals

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ABSTRACT

Here we propose a new platform for digital nucleic-acid assay based on microdroplet array. The hydrophilic-superhydrophobic property of the pseudo-paper photonic nitrocellulose substrate is employed to divide an aqueous sample containing target nucleic acids into hundreds of microdroplets for loop-mediated isothermal amplification (LAMP). The LAMP reaction leads to production of fluorescent calcein. By counting the number of microdroplets that are fluorescent, target nucleic acid (i.e. staphylococcus aureus DNA) with the copy number from 1 to 1000 can be absolutely quantified without using sophisticated microfluidic devices. The effect of fluorescent enhancement attributing to the photonic nitrocellulose can effectively amplify the detection signal and reduce the amplification time. Thus, we believe the proposed platform for digital nucleic-acid assay based on microdroplet array is promising for rapid and cost-effective bioanalytical applications.


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INTRODUCTION Digital nucleic-acid assays such as digital polymerase chain reaction (dPCR) is a range of versatile tools that enable highly sensitive, absolute quantification of nucleic acids without requirement of a standard curve.1, 2 These tools have successfully been employed in all kinds of applications such as clinical diagnostics and biological research that involves accurate quantitative detection of trace nucleic acids in complex sample matrix.3-7 In a representative digital nucleic-acid assay, the nucleic acids sample is divided into large numbers of discrete partitions, so that the nucleic acid is randomly distributed in these partitions with some containing no copy and the others containing one or more copies. Every partition serves as an independent reactor for amplification, and then the amount of partitions which contains the nucleic acid (the positive points) was obtained to calculate the total copies of nucleic acids through the following equation： λ = -ln(1 - k/n) where λ is the average copy number of nucleic acids per partition, k stands for the number of the positive points and n for the total amount of reaction partitions, and the total number of copies were estimated by multiplying λ by n.8 Other than PCR, a range of isothermal amplification methods for nucleic acids can be also utilized such as loop-mediated isothermal amplification (LAMP),9, 10 rolling circle amplification (RCA),11 multiple displacement amplification (MDA).12 By these methods, the amplification of nucleic acid is carried out at a constant temperature so that sophisticated temperature cycling equipment for PCR reaction is not required. One of the crucial steps of these digital nucleic-acid assays is how to evenly divide an aqueous sample into more than hundreds of monodisperse partitions (or microdroplets). Up to now, various approaches have been developed to realize the sample partition, which usually fall into two categories: (1) solid micro-chambers/wells and (2) water-in-oil emulsion. The first one generally 3 ACS Paragon Plus Environment


relies on a microfluidic chip containing hundreds to thousands of microchambers.13-16 When the aqueous sample flows through the chip, it fills into the microchambers which results in separated partitions. For the second category, water-in-oil emulsion is generated using a microfluidic device which leads to compartmentalization of monodisperse sample microdroplets in an oil carrier fluid.8, 17-19 Both of two types of paradigm have already been commercially available, such as Fluidigm BioMark system, Thermo Fisher Quantstudio 3D, Bio-Rad QX100/200, and Raindance Raindrop system.20 However, the conventional platforms for digital nucleic-acid assays still have some disadvantages. The fabrication of microfluidic devices relies on sophisticated micro/nanofabrication facilities and cleanroom environment. The accurate manipulation of tiny volume of aqeuous sample usually involves bulk, complicated pumping and valving systems. For the water-in-oil emulsion, it is required to stabilize the water-oil interface and prevent droplet coalescence.21-24 These issues limit the application of most conventional digital nucleic-acid assays to laboratory usage. For rapid, inexpensive and on-site bioanalysis (e.g. point-of-care testing), new platform for the digital nucleic acid assays is highly in demand. In this paper, we presented a new type of platform based on micropatterned photonic nitrocellulose substrate to divide the sample mixed with amplification reagents into microdroplet arrays for digital LAMP assay, as schemed in Figure 1. The force that drives the formation of the microdroplet is the hydrophilic-superhydrophobic interaction between the aqueous sample and the substrate, which was previously reported by Levkin and co-workers and Song et al.25-29 We demonstrate that the division of microdroplet can be accomplished on a photonic nitrocellulose substrate and used for digital nucleic-acid assays with no sophisticated microfluidic required. Furthermore, with the fluorescence enhancement effect of the photonic substrate, the detection


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signal can be further amplified so that the time required for LAMP can be shortened. As the proofof-concept analyte, SA DNA is absolutely quantified using the proposed method.



RESULTS AND DISCUSSION In this work, we fabricated a substrate with 768 (32 x 24) hydrophilic spots (Figure S1) to implement the digital nucleic acid assays based on LAMP. The hydrophobic area surrounding the spots was fabricated by laser printing of the black hydrophobic toner particles. Although the toner is hydrophobic with a water-contact-angle of 111.9° (Figure 2a), some water can still stick onto it which hinder the formation of the separated droplet arrays. In order to increase the hydrophobicity of the toner, we added superhydrophobic silica nanoparticles into the toner powder and completely mixed them into a homogeneous suspension. The wettability of the printed toner layers with different amount of silica nanoparticles (from 0 wt% to 30 wt%) was evaluated by the contact-angle measurement, as shown in Figure 2a. The water-contact-angle generally increased with increased amount of superhydrophobic silica nanoparticles. When the percentage of silica was larger than 30 wt%, the contact angle became 151.1°, which made the printing layer superhydrophobic, so that water cannot stick onto it anymore. Compared with other surface modification methods,30, 31 this inkjet printing method is rapid, cost effective and suitable for largescale fabrication. It also avoids complicated chemical processes and costly, and sometimes toxic chemical reagents. Using our method, the hydrophilic-superhydrophobic micropatterned array was fabricated by printing the superhydrophobic toner on a hydrophilic photonic nitrocellulose substrate (with a contact angle of 43.9° in Figure 2b). Then an aqueous droplet can be moved around on the substrate using a hydrophobic rod, generating microdroplets on the hydrophilic nitrocellulose spots (Figure 2c). The formation of the microdroplets was attributed to the extremely different wettability between the photonic nitrocellulose substrate that had a water contact angle of 43.9° and the superhydrophobic toner layer with a water contact angle of 151.1°.25, 32 Since the volume 6 ACS Paragon Plus Environment

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of the microdroplets determines the number of DNA copies distributed into the microdroplets, monodisperse microdroplet were preferred for accurate measurements. To evaluate the volume of each microdroplet, the side-view photograph of the microdroplet was obtained and the height of the microdroplets was measured using ImageJ software. As shown in Figure 2d, the average height of the microdroplets are around 75 μm, and relative standard deviation is around 6.7%, which is comparable with that of other digital nucleic-acid assays reported in the literature.14 As shown in Figure 3a and 3b, the fabricated photonic crystals of monodisperse silica colloid and the photonic nitrocellulose had complementary nanostructures. The periodic hexagonal arrangement of the silica nanoparticles resulted in a photonic stopband at 640 nm, as shown in Figure 3c. Complementary to the opal structure of the silica nanoparticles, the photonic nitrocellulose fabricated using the silica template had an inverse-opal structure with a photonic stopband at 549 nm (Figure 3d), which agrees with the Bragg scattering theory.33 As a consequence, the nitrocellulose substrate fabricated had a brilliant green color. Photonic crystals have been widely utilized in the enhancement of fluorescence by the slow-photon effect for ultrasensitive detections of trace analytes.34-36 We have previously demonstrated that the highest fluorescence signal amplification can be obtained by matching the stopband of the substrate with the fluorescence spectroscopy of the fluorophore, in this case, the calcein, as shown in Figure 3d. We fabricated different photonic nitrocellulose with five photonic stopbands by altering the particle size of the silica colloidal nanoparticle (i.e. 258, 276, 291, 309 and 338 nm). Figure S2 illustrated that, the calcein on the photonic nitrocellulose substrate showed a dramatically higher fluorescence intensity than the nitrocellulose without inverse-opal structure. Owing to the appropriate location between the stopband and fluorescence spectroscopy (Figure 3d), the photonic nitrocellulose with a stopband of 540 nm resulted in a 32-fold fluorescent enhancement, which



was the highest signal amplification among these substrates. Only a 2.1-fold enhancement was observed for the photonic nitrocellulose with a stopband of 507 nm. The suppression of emission intensity was due to the overlap between the stopband and the emission spectrum of calcein. Therefore, the photonic nitrocellulose with a stopband of 540 nm was utilized to perform further experiments. In a typical digital nucleic acid assay, 2.0 μL of the sample containing the target SA DNA was introduced onto micropatterned substrate and divided into microdroplets. During the LAMP reaction in an oil bath at a temperature of 65 oC, the fluorescence from each of the test spots was measured. As described in Figure 4a, The fluorescence of calcein gradually restored with time during the LAMP reaction attributing to the precipitate formed by quencher Mn2+ and pyrophosphate (PPI).37 Compared with the control experiments which was carried out without the photonic nitrocellulose (Figure 4b), the fluorescence was significantly amplified by the substrate, as shown by the larger amount of positive points in Figure 4c. Therefore, with an amplification time of about 30 min, almost all the microdroplets that had the target nucleic acid had detectable fluorescent signal. This can mainly be benefited from the fluorescence enhancement effect of the photonic nitrocellulose we fabricated, which efficiently increased the sensitivity and reduce the assay time. Most conventional LAMP reaction in a bulk solution takes at least 60 minutes to complete. In microdroplets, the reaction kinetics is faster than that of the bulk solution owing to the smaller volume of the microdroplet, which facilitated the process of heating and diffusion. Marangoni effect which is the mass transfer along an interface between two fluids due to a gradient of the surface tension can promote the mixing inside the microdroplet which can also accelerate the amplification reaction.38


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To evaluate the applicability of our method for absolute quantification of nucleic acid, a series of dilution of samples containing from 1-1000 copies SA DNA were prepared and tested. The copy number of DNA in primary sample was verified using an ultraviolet spectrophotometer by measuring absorbance of the sample at 260 and 280 nm, and then the sample was diluted to certain concentration for analysis. As shown in Figure 5a-5d, fluorescence micrographs were taken to measure the number of positive points after completion of the digital nucleic-acid assays of samples containing DNA ranging from 1 to 1000 copies. The number of positive points increased with increasing copy number of DNA in the sample. Based the amount of positive points, one can calculate the copy number of DNA in the sample by the aforementioned Poisson statistics. For testing of the four samples, 3, 26, 155 and 634 positive points were obtained from the fluorescent image, respectively, so the number of DNA copies was calculated to be 3, 26, 173 and 1340 copies. The test results showed acceptable correlation with the expected copy number (Figure 5e). Although the digital platforms cannot match the sensitivity of real-time quantitative LAMP (qLAMP) due to sample volume constraints, our method can significantly improve the sensitivity benefiting from the fluorescence enhancement of photonic nitrocellulose, and reduce the detection time. Besides, the isothermal amplification technology such as LAMP usually leads to high selectivity. These results demonstrated that our method is promising for fast, simple and costeffective digital nuclei-acid assays (Table 1).



CONCLUSION In conclusion, we have reported a new type of platform for digital nucleic-acid assays based on the hydrophilic-superhydrophobic photonic micropatterned array as the substrate. The hydrophilic-superhydrophobic micropatterned array allowed for simple and fast sample partition into hundreds of microdroplets, which eliminated the need for costly and sophisticated microfluidic devices. The photonic nitrocellulose substrate also remarkably amplified the fluorescence signal benefiting from the slow photon effect of photonic crystals, which effectively improved the sensitivity and decreased the time consumption for the test. Using our method, DNA samples with a dynamic range covered 4 orders of magnitude could be absolutely quantified within 30 minutes. These results indicated that the proposed photonic nitrocellulose arrays provide a new platform for digital nucleic-acid assays in practical applications.


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EXPERIMENTAL SECTION Chemicals and materials. The method for extraction of Staphylococcus aureus (SA) genomic DNA was according to the instructions of DNA extraction kit (DP302, TIANGEN, Beijing, China). The DNA was firstly quantified by Nano drop (ND-1000, Calibre, Beijing, China) and stored at −20 °C. The Bst DNA polymerase (M0275S) and dNTPs were purchased from New England Biolabs and Takara Biomedical Technology Co., Ltd. (Beijing, China), respectively. The amplification

primer

sets

for

the

spa

gene

of

SA

(B3:

5’-

GCTGATAACAATTTCAACAAAGAAC-3’, F3: 5’-TCATAGAAAGCATTTTGTTGTTC-3’, BIP: 5’-TTGGCTTGGGTCATCTTTTAAGCTCTATGAAATCTTGAATATGCCT-3’, FIP: 5’TTGGCTTGGGTCATCTTTTAAGCTCTATGAAATCTTGAATATGCCT-3’) were designed using LAMP Designer online program (http://www.premierbiosoft.com/isothermal/index.html) and synthesized by Sangon Biotech (Shanghai, China). Nitrocellulose was obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Acetone, dimethylformamid (DMF), trizma pre-set crystals, potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO4), ammonium sulfate ((NH4)2SO4), betaine solution, calcein, manganese(II) chloride (MnCl2) and mineral oil were purchased from Sigma-Aldrich. Superhydrophobic silica nanoparticles and monodisperse silica nanoparticles with different diameters were purchased from NanJing Nanorainbow Biotechnology Co., Ltd. All solutions were prepared with deionised water (18.0 MΩ cm, Milli-Q Gradient System, Millipore) with ultraviolet sterilization. All reagents were used as received without further purification. Fabrication of the Patterned Photonic Nitrocellulose. The photonic nitrocellulose with inverse-opal structure was fabricated by a template method previously reported with some modification. Firstly, the colloidal crystal template was prepared based on self-assembly of 11 ACS Paragon Plus Environment


monodisperse silica nanoparticles on a glass slide by doctor-blade coating. Then nitrocellulose solution prepared with acetone and DMF (1:1 v/v) was infiltrated into the nanopores of the colloid crystal by capillary force. After solidification, the glass slide was discarded, and the inverse opal photonic nitrocellulose was obtained by etching the silica nanoparticles by HF. Nitrocellulose solution was directly solidified to prepare the substrate for control experiment. The Coreldraw 12 software was utilized to design the 32 × 24 micropatterns. The diameter of the circular spot was 500 μm, and the spacing between two spots was 250 μm. Finally, the hydrophobic toner was printed on the hydrophilic photonic nitrocellulose using a laser printer (620ND, Samsung) to obtain the hydrophilic-hydrophobic micropatterns. In order to increase the hydrophobicity of the micropatterns, superhydrophobic silica nanoparticles was previously added into the commercial toner powder and they were thoroughly mixed to form a uniform suspension. The reflection spectra of the photonic nitrocellulose were recorded by a fibre optic spectrometer (QE65000, Ocean Optics, USA). Scanning electron microscopy (SEM, S-3000N, Hitachi, Japan) was used to characterize the microstructures of these materials. Digital LAMP. LAMP reaction in this work was carried out according to a previously reported method.37 The amplification mixture contained 20 mM tris-HCl (pH 8.8), 10 mM KCl, 8.0 mM MgSO4, 10 mM (NH4)2SO4, 0.10% Tween-20, 0.80 M betaine, 25 μM calcein, 0.50 mM MnCl2, 1.4 mM dNTPs, 8.0 U/μL Bst polymerase, 0.20 μM of the outer primer (F3 and B3), 1.6 μM of the inner primer (FIP and BIP) as well as the target DNA. 2.0 μL aliquot of abovementioned amplification mixture was introduced onto the surface of micropatterned photonic nitrocellulose array. A hydrophobic pipette tip was utilized to move the droplet around on the surface of the substrate, leaving an array of separated microdroplets on the hydrophilic array on the substrate. Mineral oil was utilized for sealing during the amplification at 65 oC. To avoid evaporation of the


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microdroplets, the operation was carried out at 80% relative humidity. The fluorescent images were captured by a fluorescent microscope (BX53, Olympus, Japan).



ASSOCIATED CONTENT Supporting Information Photograph of the hydrophilic-superhydrophobic micropatterned array in Figure S1, the fluorescence enhancement by different photonic nitrocellulose in Figure S2.


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ACKNOWLEDGMENTS We gratefully acknowledge financial support from Global Experts Recruitment Program of China, Innovative and Entrepreneurial Talent Recruitment Program of Jiangsu Province, State Key Project of Research and Development (2016YFF0100802), the Fundamental Research Funds for the Central Universities (2242018K41023). This work was supported by the Key Project and Open Research Fund of State Key Laboratory of Bioelectronics, Southeast University.



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35. Hou, J.; Li, M.; Song, Y. Patterned Colloidal Photonic Crystals. Angew. Chem. Int. Ed. 2018, 57, 2544-2553. 36. Hou, J.; Zhang, H.; Yang, Q.; Li, M.; Jiang, L.; Song, Y. Hydrophilic-Hydrophobic Patterned Molecularly Imprinted Photonic Crystal Sensors for High-Sensitive Colorimetric Detection of Tetracycline. Small 2015, 11, 2738-2742. 37. Tomita, N.; Mori, Y.; Kanda, H.; Notomi, T. Loop-Mediated Isothermal Amplification (LAMP) of Gene Sequences and Simple Visual Detection of Products. Nat. Protoc. 2008, 3, 877. 38. Wegener, M.; Paschedag, A. R. Mass Transfer Enhancement at Deformable Droplets due to Marangoni Convection. Int. J. Multiphase Flow 2011, 37, 76-83. 39. Craw, P.; Balachandran, W. Isothermal Nucleic Acid Amplification Technologies for Point-of-Care Diagnostics: A Critical Review. Lab Chip 2012, 12, 2469-2486. 40. Nixon, G.; Garson, J. A.; Grant, P.; Nastouli, E.; Foy, C. A.; Huggett, J. F. Comparative Study of Sensitivity, Linearity, and Resistance to Inhibition of Digital and Nondigital Polymerase Chain Reaction and Loop Mediated Isothermal Amplification Assays for Quantification of Human Cytomegalovirus. Anal. Chem. 2014, 86, 4387-4394.



FIGURES

Figure 1. a) Scheme of the fabrication of the micropatterned photonic nitrocellulose arrays. b) Schematics of the formation of microdroplets containing DNA copies on the hydrophilicsuperhydrophobic substrate for the LAMP-based quantification.


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Figure 2. a) Water contact angles measured on the toner layer printed on the photonic nitrocellulose substrate. Diﬀerent amounts of superhydrophobic silica were added into the toner for increase the hydrophobicity. b) Photographs of the formed microdroplets on the photonic nitrocellulose array. The contact angle is indicated. Scale bar: 100 μm. c) A photograph of the formation of microdroplets on the nitrocellulose array when the sample containing DNA target was introduced on the hydrophilic-superhydrophobic substrate and moved by a hydrophobic bar. Scale bar: 2.0 mm. d) Distribution of the height of all microdroplets formed on the substrate.



Figure 3. a-b) SEM images of the colloidal-crystal template (a) and corresponding photonic nitrocellulose (b). Scale bar: 500 nm. c-d) Reflectance spectra of the silica template (c) and the corresponding photonic nitrocellulose (d). The dashed lines indicated the location of maximum excitation (Ex) and emission (Em) wavelength of calcein.


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Figure 4. a, b) Fluorescence micrographs of substrate after completion of the digital nucleic-acid assays in the microdroplet arrays with (row a) and without (row b) photonic nitrocellulose (PhNC). The sample tested contained DNA of 1000 copies. The micrographs were captured at 20 (I), 30 (II), 40 (III), 50 (IV) and 60 minutes (V), respectively. c) Positive spots in the microdroplet array as a function of reaction time.



Figure 5. a-d). Fluorescence micrographs of substrate after completion of the digital nucleic-acid assays of samples containing DNA ranging from 1 to 1000 copies. e) The copy number of the DNA calculated from the number of positive spots versus the predicted values which was verified using UV-vis measurement. The error bars represent standard deviation for three replicated measurements.


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Our method

dLAMP (commercial)

dLAMP

qLAMP

Detection signal

Fluorescence

Fluorescence

Colorimetric dye

Fluorescence

Sensitivity

~3 copies

~5 copies

~8 copies

1-2 copies

Selectivity

high

high

high

high

Dynamic range

4 orders of magnitude

2 - 6 orders of magnitude

N/A

> 9 orders of magnitude

Optimized time (min)

30

60

50

60

Partition volume (nL)

2.87

0.004~0.85

15

N/A

Printer

Commercial equipment

lithography machine

Commercial equipment

~0.15K

~45K - 250K

~160K

~25K - 50K

~0.7

5 - 400

N/A

2

Instrument Cost/sample ($)

Table 1. Comparison of our method and other quantification of nucleic acids based on LAMP.7, 20,39,40 Specifications

not available are marked with N/A.



For Table of Contents Only


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Generating Microdroplet Array on Photonic Pseudo-Paper for Absolute

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