Visualized Quantitation of Trace Nucleic Acids Based on Coffee-Ring

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Visualized Quantitation of Trace Nucleic Acids Based on Coffee-Ring Effect on Colloid-Crystal Substrate Dagan Zhang, Bingbing Gao, Chao Zhao, and Hong Liu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03609 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Visualized Quantitation of Trace Nucleic Acids Based on Coffee-Ring Effect on Colloid-Crystal Substrate Dagan Zhang, Bingbing Gao, Chao Zhao* and Hong Liu* † State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China. ABSTRACT: We report a visualized quantitative detection method for nucleic-acid amplification tests based on coffee-ring effect on colloid-crystal substrate. The solution for loop-mediated isothermal amplification (LAMP) of DNA is dropcast on a colloid-crystal surface. After complete drying, a coffee ring containing the LAMP byproduct (i.e. magnesium pyrophosphate) is formed, and it is found that the width of the coffee ring is linearly correlated to the logarithm of the original DNA concentration before the isothermal amplification. Importantly, compared with other substrates, we found that the colloidalcrystal substrate is an appropriate substrate for carrying out the assay of high sensitivity. Based on these findings, we develop a coffee ring-based assay for quantitative readout of trace DNA in sample. The assay requires 0.50 µL sample and is completed in 5 min in a homemade chamber with constant humidity. Semi-quantitative detection of trace DNA is performed using naked eyes. With the use of a smartphone, the DNA in a sample can be quantitatively detected with a limit-of-detection of 20 copies. The detection of specific nucleic acid targets plays a significant role in biomedical research, diagnostics, food safety, forensic science and so forth.1-5 Although various methods and instruments have been developed, point-ofcare testing (POCT) of nucleic acids suffers from low sensitivity and thus high detection limit compared with other conventional laboratory-based testing. For some critical applications, the amount of target nucleic acid is extremely small compared to other components in the sample. For example, as a promising target for early diagnostics of cancer, circulating tumor cells are very rare in blood, usually in the range of 1‒100 cells/mL.6-9 For another promising candidate for cancer diagnostics (i.e. MicroRNA), the concentration is as low as a few copies per cell.10 Therefore, a range of nucleic acid amplification methods such as PCR, recombinase polymerase amplification (RPA), strand displacement amplification (SDA), rolling circle amplification technology (RCA), loop mediated isothermal amplification (LAMP) have been used for enhancement of the signal.11 LAMP technique is first reported by Notomi et al. as a novel nucleic acid amplification method, which is a popular technique for the detection of various biomolecules owing to its simplicity and high sensitivity. 11 LAMP is a convenient and effective nucleic acid amplification method, 12 which relies on strand displacement activity of Bst DNA polymerase and specially designed two or three sets of primers to amplify target nucleic acid under isothermal conditions (60-65 oC) without thermal cycler in less than 1.0 h. 13 In addition, the LAMP has many advantages, including simple operation, low cost and only need simple tools, such as a bath or a heating block. During the process, pyrophosphate (PPi) is released as a byproduct, and the amount of PPi produced is proportional to the amount of original nucleic acid in the

Scheme 1.Schematic illustration showing the coffee-ring based assay for quantification of nucleic-acid amplification tests on colloid crystal of SiO2 nanoparticles. sample. The PPi further reacts with magnesium ion to form magnesium pyrophosphate precipitate, which are related to the degree of turbidity.11 As a powerful nucleic acid amplification method with high specificity and sensitivity, LAMP has found many applica tions in detection of microRNA,14specific DNA sequence,15 single nucleotide polymorphisms,16 viruses17 and a series of pathogenic microorganisms such as Escherichia coli,18 Salmonella,19 Yersinia pseudotuberculosis 20and biomarkers of interest.2 Compared with conventional amplification methods such as PCR, LAMP simplifies the amplification process by eliminating the thermocycling steps.11 It also reduces equipment costs, and improves the sensitivity for biochemical analysis.11 Therefore, quite a lot of efforts have been made to employ LAMP in point-of-care testing (POCT) to decrease the limit of detection (LOD).21-23 However, most of the methods for the detection of LAMPbased assays are based on turbidimetric,18 colorimetric,24,25 fluorescent,26 electrochemical,27 chemiluminescent measurement.28 The stained DNA can be qualitatively visualized by naked eyes based on the color change of the dye. But for quantitative measurements, sophisticated instruments are usually required.29 For turbidity

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measurements, which detects the magnesium pyrophosphate as a white-precipitate byproduct of LAMP, however, it requires an expensive and complicated turbidimeter, which is not suitable for point-of-care testing.25 Although fluorescent measurements bring about sensitive and quantitative results, the primers in excess result in strong background fluorescence. The separation of the amplified nucleic acid using gel electrophoresis involves highly toxic reagents and highly-trained personal, which restricted their widespread application.30 Therefore, development of new readout method that is more suitable for point-of-care applications is highly in demand. The coffee-ring effect (CRE) is a phenomenon that can occur with a drying drop on a solid surface. The physics of the CRE was previously described by Deegan and coworkers. 31 The higher evaporation rate at the threephase contact line induces a capillary flow that transports particles in solution to the periphery of the drop. The radial flow leads to formation of a characteristic ring pattern on the substrate after complete evaporation of the solvent. CRE has attracted extensive attention and found many interesting applications such as inkjet printing,32 nanochromatography,33 mass spectroscopy,34 surfaceenhanced raman spectroscopy,35 preconcentration for biosensing36-38 and diagnosis of diseases.39-41 We have previously reported a quantitative analytical method using the naked eye that is based on the CRE on paper. By the CRE which converts a colored solution to stains on the paper. The width of the colored stain was linearly correlated to the logarithm of concentration of the analyte.42,43 However, due to the randomly-structured micropores of the cellulose paper used in this work, the capillary flow on the paper was not quite even. The uneven flow on the paper can result in blurred color boundary which makes accurate quantitative measurement rather challenging. In this work, we use a colloid crystal membrane as the substrate for carrying out nucleic-acid amplification test based on the CRE. The colloid crystal is made of selfassembled monodisperse SiO2 nanoparticles.44 The nanoparticles are hexagonally closed packed so that the pores in between the nanoparticles are highly ordered (Figure 1). We hypothesize that the capillary flow on this highly ordered substrate can be more uniform than that on the cellulose paper. The uniform convection flow in the droplet can bring about coffee ring with clearer edge which enables highly sensitive detection. As a proof-of-concept target, Salmonella is quantitatively analyzed using our method. Salmonella is an important pathogen which infects many people and has become a serious problem so that the detection of Salmonella is an urgent need. For the coffee-ring based assay, we developed a protocol for LAMP amplification of Salmonella in solution. The Salmonella genomic is 4.6×106 bp, the assay of targeting bcfD gene was one conserved fimbrial operon gene from Salmonella.

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Figure 1. (a) Scanning electron micrographs of the colloid crystal of SiO2 nanoparticles with a diameter of 280 nm. (b) A photograph of the colloid crystal membrane assembled using the SiO2 nanoparticles. (c) Reflectance spectra of the colloid crystal of SiO2 nanoparticles. The concentration of the purified target DNA was measured based on the optical density (OD) at 260 nm using NanoDrop-2000 apparatus (Thermo Scientific), and the original copy number of genomic DNA was calculated to be 107 copies/l. The DNA sample was diluted from 107 copies/L to 102 copies/L using 0.010 M TE buffer (pH 8.0), and a sample containing no DNA was also prepared as the control. The reaction mixture containing 2.0 µL of the sample and 23 µL of the amplification reagents was incubated at 65 °C for 60 min to amplify the target DNA, as shown in Figure S1 (detailed experimental procedure was available in Supporting Information). The amplified LAMP products were then analyzed by electrophoresis using a 2.0% agarose gel. As shown in Figure S1, the DNA of Salmonella was amplified, so this procedure was used for the coffeering detection. To prepare the colloid crystal membrane, a 60 µL aliquot of ethanol suspension containing 20% (w/v) monodisperse SiO2 nanoparticles was dropcast onto a plasma-treated glass slide. An automatic coater was used for blade-coating of the suspension on the glass slide. After complete evaporation and self-assembly at a room temperature of 25 °C, a hexagonal close-packed colloid crystal was obtained, the nanoparticles form highly-ordered nanostructure which reduces the surface roughness and can support more uniform capillary flow than other surface (Figure 1a).44 As shown in Figure 1b, the colloid crystal membrane showed a red color, which arises from the periodic nanostructure of the highly-ordered SiO2 nanoparticles that selectively reflect photons of certain wavelength at 640 nm (Figure 1c). To investigate the formation of coffee ring on different substrates, a 0.50 µL aliquot of solution containing DNA at 102 copies/L after the LAMP reaction was dropcast on to the substrates (i.e. glass slide, PET membrane, nitrocellulose (NC) membrane, filter paper, substrates fabricated

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Figure 2. Photographs of a drop of 0.50 µL aliquot of solution containing DNA at 102 copies/L after the LAMP reaction before and after drying for 5.0 min on different substrates including glass side, PET membrance, NC membrance, paper, disordered and ordered SiO2 colloid substrates. (scale bar :5 mm). by self-assembly of polydisperse and monodisperse SiO2 nanoparticles respectively), and allowed to dry (Figure 2). Since both the glass slide and the PET membrane were not porous, a liquid drop formed on the substrate. After complete drying magnesium pyrophosphate precipitate was observed on the substrate. For the other porous cellulose-based substrate, the liquid wicked into the pores by capillary force. After complete drying, the magnesium pyrophosphate precipitate was not observed because of the white background color of the paper substrates. Finally, for the membrane of SiO2 nanoparticles with disordered and ordered structures, a coffee ring was formed on the substrate after drying. However, ring cannot clearly distinguish for the disorder structure membrane. For the order colloidal crystal substrate, because of the structure color of the colloid crystal substrate fabricated using monodisperse SiO2 nanoparticles, the contrast between the ring and the background was more obvious than that of the other substrate. Note that the color edge was also rather clearer, which enables more accurate quantitative measurements compared with our previous reports.42 The evaporation of water was significantly influenced by humidity of the ambient environment. 45 We investigated the influence of humidity on the formation of the coffee ring, and the results were shown in Figure 3. Quite a few parameters of the coffee ring were affected by the relative humidity of the environment such as the inner diameter, the outer diameter (Figure 3a), and the time required for the formation of the coffee ring (Figure 3b). To ensure the reproducibility of the coffee-ring based tests, a home-made dry chamber which was sealed and filled with silica desiccant (Figure 3c) was employed to provide a constant humidity.39 As shown in Figure 3d, the relative humidity in the chamber decreased with time after loading the desiccant and it reached a plateau (~17%) at about 70 min. Note that a small window was available on the chamber for introduction of the test substrate after LAMP reaction, and the change of the relative humidity owing to the opening of the window was negligible.

Figure 3. (a) Photographs of 0.50 μL aliquot of the solution containing DNA at 104 copies/L after the LAMP reaction and drying for 5.0 min on the colloid crystal substrate at a relative humidity of 80%, 50%, 17%, respectively (scale bar: 4 mm). (b) The time required for 0.50 μL aliquot of the solution containing DNA at 104 copies/L after the LAMP reaction to completely dry under different relative humidity. (c) A photograph of the homemade humidity chamber filled with silica desiccants. (d) Relative humidity inside the sealed chamber as a function of time. To demonstrate the applicability of coffee-ring effect on the colloidal crystal substrate to quantitative readout of the LAMP-based nucleic acid test, a few colloidal crystal substrates were prepared (Figure 4a). Next, 0.50 μL aliquot of the solution after the LAMP reaction were dropcast onto the colloid crystal substrate. The substrate was placed in the dry chamber to dry and the solution complete dried after 5.0 min. Finally, the coffee ring was formed on the substrate. The scanning electron micrograph of the coffee ring was shown in Figure 4b and 4c. As previously mentioned, the evaporation of the liquid droplet induced a capillary flow which brings particles (i.e. magnesium pyrophosphate precipitate) in the droplet to move from the center to the edge. With the solvent evaporation, the particles gradually accumulated at the edge of the ring.

Figure 4. (a) Scanning electron micrographs of the colloid crystal of SiO2 nanoparticles with a diameter of 280 nm. (b) Scanning electron micrographs of LAMP products for the DNA at 104 copies/L and drying for 5.0 min on the colloid crystal substrate to form coffee-ring (scale bar :10 um). (c) Scanning electron micrographs of the LAMP products (scale bar :2 um).

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required no complex instruments and was less costly as compared to previous approaches. We believe that this method can be further used for the development of various kinds of diseases with the naked eye, so it is promising for point-of-care diagnosis under resource-limited conditions.

ASSOCIATED CONTENT Supporting Information Information about chemicals, materials, experimental procedures, and additional Figures. This material is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Figure 5. (a) Photographs of 0.50 μL aliquot of the solution containing DNA at different copies after the LAMP reaction and drying for 5.0min on the colloid crystal substrate (scale bar: 5 mm). The concentration of DNA is indicated. (b) Width of the colored ring D as a function of DNA concentration. (c) Width as a function of logarithm of the DNA concentration. The width was measured vertically and horizontally, and the average is shown in the plot. The error bars represent standard deviation for three replicated measurements. Similar to the results we have previously reported,42,43 it was found that the width of the coffee ring is correlated to the original concentration of the target DNA in the testing sample, as shown in Figure 5a. A camera was used to obtain optical photographs of the coffee ring so that the diameters of these coffee rings were measured. As shown in Figure 5a, the inner diameters of these coffee rings decreased with increasing DNA copies. This was reasonable because magnesium pyrophosphate particles produced from the LAMP reaction is proportional to the original DNA copies in the sample. When the magnesium pyrophosphate particles accumulated at the edge of the liquid droplet, they slowed down the capillary flow so that the coffee ring tended to become wider with increasing amount of particles in the solution. For quantitative detection, we measured the inner diameter of the ring, which was calculated as D shown in Figure 5b. The width of the ring was linearly correlated to logarithm of DNA concentration from 107 to 102 copies/L (Figure 5c). The detection limit, calculated as three times the standard deviation of the blank divided by the slope, was 20 copies/L. For rapid detection on site, we also developed a smartphone App for measuring the width of the coffee ring. As shown in Figure S2, the DNA in a sample can be quantitatively detected with just the smartphone. To summarize, we developed a novel method for quantitative detection of nucleic-acid amplification tests with naked-eye based on coffee-ring effect on colloidcrystal substrate. We dropcast the solution for LAMP of DNA on a colloid-crystal surface and found that a coffee ring containing the LAMP byproducts (i.e. magnesium pyrophosphate) was formed after complete drying, and the width of the coffee ring was linearly correlated to the logarithm of the original DNA concentration before the isothermal amplification. The detection only required 0.50 µL sample and was completed in 5 min. This method

Corresponding Author [email protected] and [email protected]

Notes

The authors declare no competing financial interests.

ACKNOWLEDGMENT We gratefully acknowledge financial support from Chinese Recruitment Program of Global Experts, 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) and Jiangsu Provincial Medical Youth Talent (QNRC2016540). This work is supported by the Key Project and Open Research Fund of State Key Laboratory of Bioelectronics, Southeast University.

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