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Article Cite This: ACS Omega 2019, 4, 1611−1616
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Mix-and-Read No-Wash Fluorescence DNA Sensing System Using Graphene Oxide: Analytical Performance of Fresh Versus Aged Dispersions C. Lorena Manzanares Palenzuela, Amir Masoud Pourrahimi, Zdeněk Sofer, and Martin Pumera* Center for the Advanced Functional Nanorobots, Department of Inorganic Chemistry, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague 6, Czech Republic
ACS Omega 2019.4:1611-1616. Downloaded from pubs.acs.org by 146.185.200.69 on 01/20/19. For personal use only.
S Supporting Information *
ABSTRACT: Simple and sensitive assays for DNA detection still represent a highly pursued research area with important implications in biomedical-related sciences. Graphene oxide (GO) is a highly efficient quenching platform for fluorophoretagged DNA, which is why its use for fluorescent sensing has been widespread over the past decade. GO-based biosensing systems frequently rely upon the isolation of biomolecule− material complexes prior to detection via hybridization-induced desorption of the fluorescent dye. Simple mix-and-read detection formats that do not require purification/isolation/ wash steps are envisioned as promising schemes for decentralized analysis, with potential for commercial scalability. For GO-based mix-and-read assays, the aging process of the quenching material in aqueous media can be a crucial parameter affecting the analytical performance, which has so far not been addressed in the literature. To get this goal, top−down characterization microstructures to atomic levels is needed. Herein, we revisit GO as a well-known quenching system, aiming at a centrifugation-free, mix-and-read, no-wash format, toward the detection of an apolipoprotein-E-encoding DNA sequence as a model analyte. We look into the progression of GO aging in water medium through a top−down characterization and investigate the analytical performance of fresh versus aged dispersions in terms of hybridization-based detection. We found that aged GO, while still retaining a high quenching efficiency, undergoes morphological changes over time with concomitant detrimental effects on its analytical performance toward DNA detection.
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nanomaterials,7−10 especially carbon-based, has been exploited in the last decade for the development of fast detection assays with minimal protocol steps.5,6 Graphene, as the basic block of carbon allotropes, exhibits distinct properties, such as its unusual structural characteristics and remarkable electronic, mechanical, and thermal properties.11 Almost one decade ago, the ability of water-dispersible graphene oxide (GO) as a platform for detecting DNA using a Förster resonance energy transfer (FRET) approach was first demonstrated.12 Ever since, applications in biosensing and live cell bioimaging10,13 and, most recently, studies on DNA translocation through graphene nanopores for sequencing14 still continue to be extensively addressed. A quick literature search retrieves a myriad of reports only on the biosensing field, a big fraction of which is taken up by FRET biosensors. Even though new layered materials, namely graphene-like two-dimensional structures, are being evaluated for FRET-based applications,7,10,13 the quenching efficiency of
INTRODUCTION Detecting biomolecules has been a highly pursued research topic for decades as it represents the cornerstone of biomedical-related sciences. The field of molecular diagnostics, that is, sequence-specific DNA detection, has evolved tremendously with the discovery of real-time polymerase chain reaction (PCR) in the nineties. Even though this fluorescence-based technique is considered the gold standard for DNA analysis and gene expression studies in research and clinical laboratories to this date, researchers are incessantly working on developing portable analytical devices to minimize the inherent delays associated with routine laboratory-based diagnostics.1−3 By providing faster results, these portable assays may influence early treatment decisions. Fluorescence-based “mix-and-read” (also referred to as “nowash”) assays are appealing for these purposes, given the technique’s superior sensitivity compared to colorimetric/ visual methods as well as its simplicity in terms of execution, which favors its scalability as kit-based assays.4−6 Such systems can be beneficial for low-resource settings as well as bedside diagnostics without the need of sample transportation and specialized staff. In this sense, DNA interaction with micro/ © 2019 American Chemical Society
Received: October 19, 2018 Accepted: November 16, 2018 Published: January 18, 2019 1611
DOI: 10.1021/acsomega.8b02885 ACS Omega 2019, 4, 1611−1616
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Figure 1. Fresh and aged GO characterization: SEM images of (a) powder state and (b,c) fresh and 1 week-aged GO deposited on a Si wafer, (d− f) SEM images at different magnifications together with EDS elemental distribution of carbon and oxygen for 2 months-aged GO deposited on a Si wafer, (g,h) high-resolution XPS of the C 1s region for the fresh and 2 months-aged GO, and (i) Raman spectrum for the fresh and 2 months-aged GO.
graphene-based materials is still practically unbeatable.15 Recent reports have taken the concept of GO−DNA interactions from synthetic oligonucleotides to real samples by coupling PCR amplification with this material’s adsorptive and fluorescence quenching properties.16−18 Yet, an important but often overlooked issue in the DNA detection field is how the aging process of GO in terms of both chemical and morphological changes can affect the analytical performance of the FRET system. GO’s physical and chemical properties are prone to be altered by aging effects, which are known to affect the binding of biomolecules.19 In this work, we undertook DNA detection in a simple centrifugation-free, mix-and-read scheme, investigating the analytical properties of fresh versus aged dispersions. As a model analyte, we detect a short DNA sequence encoding for apolipoprotein E, a genetic biomarker associated with hypertension20 and Alzheimer.21,22
Figure 2. High-resolution SEM images at different magnifications together with EDX elemental distribution for GO deposited on the Si wafer; (a,b) fresh and (c,d) 1 week-aged. All scale bars are 1 μm.
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RESULTS AND DISCUSSION Figure 1 presents the nano/microstructure of drop-casted GO with different aging times in water medium from aqueous suspension, as revealed by scanning electron microscopy (SEM)/energy-dispersive spectrometry (EDS), X-ray photoelectron spectroscopy (XPS), and Raman characterizations. Figure 1a shows the micrograph of primary GO powder that has relatively flat sheets with a wrinkle appearance, which is also present after its aqueous suspension drop-casting on a Si wafer, as presented in Figures 1b and 2a. The scanning micrographs of drop-casted fresh and 1 week-aged samples, presented in Figure 1b,c, show that the GO formed entirely a thin layer with a fine nano-sized thickness together with micrometer-sized species, where the appearance of microparticulates per surface area increased through aging within 1 week. To gain more information about the morphology of these microparticulates through 1 week of aging in water medium, the high-resolution SEM images coupled with EDS elemental mapping are shown in Figure 2. Through the aging of GO in water medium, the wrinkling and folding on the surface of the microparticulates increased, whereas their sizes
reduced. After 2 months of aging, these species were evolved into smaller size microparticles with well-defined oval shapes, which strongly adhered to GO thin-layer sheets (Figures 1d−f). The atomic force microscopy (AFM) image shows the topography of casted GO, where the particles’ dimensions ranged from ∼70 to 270 nm in height and ∼1−1.5 μm in width; see Figure S1. Dynamic light scattering (DLS) measurements (Figure S2) revealed two peaks of 462.4 and 2692 nm of hydrodynamic size each (polydispersity index: 0.361). These results suggest that the two morphologies also coexist in water medium, that is, oval-shaped microparticles coexisting with nanosheets. The surface of the micrometersized particles contained less carbon content than the formed thin layer, as revealed by EDS elemental mapping (shown in Figure 1e). It should be noted that the interaction volume for elemental mapping in the EDS measurement is less than the height of the particles, and therefore the surface of the particles was subjected to elemental analysis. 1612
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The XPS-derived carbon/oxygen (C/O) ratios were 2.9 and 1.5 for the fresh and 2 months aged GO samples, respectively. To gain more information about the functional groups associated with carbon, the deconvoluted high-resolution C 1s XPS spectra of fresh and aged GO samples are shown in Figure 1g,h, respectively. GO is a thin sheet (with atomic-level thickness) carbon covalently bonded with functional oxidized groups containing sp2 (fresh: 25.62% and aged: 27.58%) and sp3 hybridized carbon atoms (fresh: 19.30% and aged: 21.81%). Together with the sp2 graphitic component and sp3 hybridization, deconvolution of the C 1s peak in GO presents the presence of oxygen-carrying functional groups. The component at 286.95 eV, assigned to C atoms directly bonded to oxygen with hydroxyl and epoxy configurations has high contributions: 52.36% for fresh and 44% for aged, whereas the component at 288.43 eV is assigned to carbonyl groups (fresh: 2.7 and aged: 6.62% of total carbon). The Raman spectra of GO in Figure 1i show two most significant peaks, namely, a G band at ∼1586 cm−1 and a D band centered at ∼1354 cm−1, which are respectively related to the in-plane motion of the carbon atoms and the presence of disorder and defects in the sheet. The D/G intensity ratio (ID/IG) is therefore used to assess the level of disorder in GO, which is calculated as ca. 0.95 for both aged and fresh samples being similar to the values reported in the literature.23 Therefore, it is hereby shown that the physical structure and morphology of GO severely transformed from nanosheets with a wrinkled and folded appearance into heterostructure nanosheets containing oxygenenriched microparticulates, whereas the overall surface chemistry of GO changed slightly through 2 months of aging in water medium. There are few articles that explain the degradation mechanism of GO considering both morphology and surface chemistry through aging in water medium. It was shown that GO aqueous suspension originally contains almost no preexisting acidic functional groups but may generate them through interaction with water in longer aging times.24 The reaction with water results in the degradation of GO flakes and may lead to C−C bond cleavage and the generation of protons (humic acid-like structures) via formation of an electrical double layer at the GO/water interface. In another study, it was shown that the carboxyl groups are protonated such that the GO sheets become less hydrophilic and form aggregates at low pH,25 which may promote the number of wrinkles and foldings on the surface of GO through aging. The degradation of other carbon-based structures such as carbon black has been studied at low pH, and it was suggested that the produced acid structure accumulated on the surface accessible parts (here the wrinkle spots).26 In our case, it is suggested that the produced protons in the GO aqueous suspension attack the wrinkle spots as the nucleation points for the formation of microparticulates. We assessed DNA binding to fresh and aged GO by means of fluorescence measurements. Figure 3 shows the kinetic profiling of fluorescein amidite (FAM)-ssDNA adsorption onto both platforms. The quenching efficiency of fresh and aged GO, calculated from the time constant values of both exponential decays, that is, t1, were 98.4 and 99.2%, respectively. The data collected up to 10 min were fitted with double exponential functions. The higher quenching efficiency and faster decay observed for the aged dispersion could be assigned to hydrogen-bonding contributions by the oxygen-rich microparticles. Xu et al.27 recently studied the cooperation of hydrogen bonding and π-stacking in ssDNA
Figure 3. Kinetic profiling of GO−FAM-ssDNA interaction measured at an excitation wavelength of 490 nm. Emission data were recorded at 520 nm every second during 30 min. Double exponential fitting was performed for the three systems from 0 to 600 s. The decay time constants (t1), denoted as t, are shown with their standard deviation values. An enlarged region (0−40 s) is displayed above, showing the moment of GO addition (fresh and aged GO dispersions are represented by blue and red dots, respectively; FAM-ssDNA is shown as black dots).
adsorption on GO using molecular dynamics simulation. The authors revealed that ssDNA could be adsorbed on a GO surface, with preferential binding to the oxidized rather than to the unoxidized region of the material, first by the hydrogenbonding interaction, followed by π−π stacking interactions. According to the authors, it takes 0.17 ns to form a hydrogen bond between the ssDNA’s phosphate backbone and the GO surface, whereas it takes 0.986 ns for the formation of stable π−π stacking between the aromatic rings of the nucleobases and those of the GO surface. The next set of experiments were aimed at performing the assay in a mix-and-read, no-wash format, by mixing each GO dispersion with either ssDNA or dsDNA and including one double-stranded structure containing one mismatched base. After 1 h of GO−FAM-ssDNA incubation time, Figure 4 shows that FAM quenching is close to 100% for both systems, correlating with the kinetically derived efficiencies. When dsDNA is added to the fresh material, the fluorescence retained ∼10% of that of the original FAM-ssDNA. This is due to the differential affinity of GO toward ssDNA and dsDNA, the latter being weaker as the nucleobases are stacked within the double helix and unavailable for interaction with the GO network, whereas the aged GO−dsDNA system showed a striking fluorescence decrease, that is, almost 1 order of magnitude less intensity than fresh GO−dsDNA and 2 orders of magnitude less than free FAM-ssDNA. The detection of one-base mismatch was also assessed: the fresh GO dispersion was able to discriminate it (∼30% of signal decrease compared to the fully complementary target DNA), contrary to the aged one, which showed a negligible signal difference compared to the target sequence. These findings suggest that dsDNA strands might be adsorbed to some extent onto the oxygenenriched particulates of the aged GO, possibly via the phosphate backbone. Because of the higher rigidity of the 1613
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Figure 4. Representation of fresh vs aged systems with the collected emission spectra for FAM-ssDNA, GO−FAM-ssDNA, GO−dsDNA (fully complementary DNA target), and GO−1 mismatch, measured at an excitation wavelength of 490 nm. Three-dimensional SEM representations of each system are shown on a 30 μm × 30 μm scale. The concentration of DNA target and 1-mismatch was 50 nM, forming equimolar complexes with FAM-ssDNA.
network that greatly compromises the system’s analytical parameters. Aged GO resulted in an unsuitable platform for DNA detection because of its inability to differentiate between small differences in DNA target concentration, that is, sensitivity, and signal discrimination with one-mismatched sequences, that is, specificity.
double strands compared to single strands, the adsorption of the former is still less than that of ssDNA.28 Figure 5 shows the analytical performance of both systems in terms of sensitivity. It is clear from Figure 5a,b that the analytical performance of the fresh GO dispersion is superior to that of the aged material. A linear range spanning 1 order of magnitude (see semilogarithmic fitting in Figure 5b) with a low detectable level of 5 nM was obtained for the fresh GO system (R2 > 0.99). It should be noted that this no-wash assay performed at room temperature exhibited comparable or higher sensitivity than previously reported methods where temperature control,29 hybridization enhancers,30 and/or centrifugation-assisted separation of the complexes were implemented.31 The aged GO system, on the other hand, showed a significant loss of sensitivity. As previously stated, this difference can be assigned to strong hydrogen-binding contributions by the oxygen-enriched heterostructures in aged GO (Figure 1d−f), prone to adsorb both ssDNA and dsDNA, ultimately resulting in this system’s inability to differentiate between different concentration levels of target DNA or to discriminate one single mismatch (Figure 5c).
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EXPERIMENTAL SECTION
Chemicals. Pure graphite microparticles (2−15 μm, 99.9995%) were obtained from Alfa Aesar, Germany. Sulphuric acid, phosphoric acid, potassium permanganate, and hydrogen peroxide were acquired from Penta, Czech Republic. Magnesium chloride hexahydrate, tris(hydroxymethyl)aminomethane, sodium chloride, hydrochloric acid, and DNA oligonucleotides were purchased from Sigma-Aldrich (UK). The oligonucleotide sequences are shown in the Supporting Information (Table S1). Ultrapure water used in this study was obtained from a Milli-Q ion exchange column (Millipore) with a resistivity of 18.2 MΩ cm at 25 °C. For DNA−material interaction studies, 0.125 M Tris-HCl buffer solution (pH 7.2, 0.25 M NaCl and 0.025 M MgCl2) was used. GO Synthesis. GO was prepared according to the modified Hummers method, as previously described.32 Briefly, graphite and potassium permanganate were added to a 9:1 mixture of concentrated sulphuric acid and phosphoric acid and cooled under 0 °C, followed by slowly heating to 50 °C and stirring during 12 h. After cooling and removing the permanganate ions and manganese dioxide excess with hydrogen peroxide, the obtained GO was purified by centrifugation cycles. The resulting GO slurry was dried in a vacuum oven. GO dispersions (1 mg mL−1) were prepared in water and left at room temperature for aging studies, that is, dispersions were characterized in terms of morphology at different times: they
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CONCLUSIONS We have evaluated the analytical performance of a fluorescent sensing system based on a mix-and-read DNA detection assay using GO as the quenching platform. We addressed the unexplored task of assessing the effect of the GO aging process in this type of widespread assay. More specifically, we addressed a comparison study between fresh and aged GO dispersions in water medium, in terms of morphological and chemical changes and their impact on linearity and single-base mismatch discrimination. We found that GO aging progresses with the formation of an oxygen-enriched microparticulated 1614
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(Omicron Nanotechnology, Taunusstein, Germany) instrument with a monochromatic aluminium X-ray radiation source (1486.7 eV). An electron gun was used to eliminate sample charging during the measurement (1−5 V). Raman spectroscopy was performed with an inVia Raman microscope (Renishaw, England) in backscattering geometry with a charge-coupled device detector. DPSS laser (532 nm, 50 mW) with an applied power of 0.025 mW and a 50× magnification objective was used for the measurement. AFM measurements were carried out on an Ntegra Spectra from NT-MDT. The surface scans were performed in the tapping (semicontact) mode using drop-casted dispersions on a freshly cleaved mica substrate. Gwyddion software was used for image analysis. The measurement was performed under ambient conditions with a scan rate of 1 Hz and a scan line of 512. DLS was performed using a Zetasizer Nano ZS (Malvern, England) analyzer. The measurements were done at room temperature (20 °C) with diluted dispersions using polystyrene cuvettes. DNA Adsorption and Detection Assays. Aqueous 1 mg mL−1 GO dispersions were let to naturally sediment to retrieve the fine supernatant particles for the sensing assay. These fine particles were mixed with FAM-labeled single-stranded oligonucleotides (FAM-ssDNA) to a final concentration of 50 μg mL−1 and 50 nM separately in Tris-HCl buffer (final concentration set to: 50 mM Tris-HCl, 100 mM NaCl, and 10 mM MgCl2) and incubated for 1 h at room temperature. Separately, DNA hybridization studies were carried out by incubating increasing amounts of target DNA with FAMssDNA for 1 h at room temperature. A single-base mismatched sequence was also hybridized separately with FAM-ssDNA. These hybrids were then transferred to the GO dispersions, keeping the abovementioned conditions of ionic strength and pH. Fluorescence spectra were recorded at an excitation wavelength of 490 nm using Fluorolog Extreme (Horiba, France) equipped with a Xe lamp (450 W) and a double excitation monochromator. A monochromator iHR320 with a thermoelectrically cooled PMT detector was used for the measurement of emission spectra.
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ASSOCIATED CONTENT
S Supporting Information *
Figure 5. Analytical performance of fresh and aged GO dispersions (blue and red, respectively) toward DNA detection and single-base mismatch discrimination in a mix-and-read, no-wash assay format: (a) fluorescence intensity (emission wavelength: 520 nm; excitation wavelength: 490 nm) vs target DNA concentration; (b) semilogarithmic fitting of the 1−50 nM range; (c) mismatch discrimination. The error bars are the standard deviation of three separate measurements.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02885. Oligonucleotide sequences with accession number; AFM image of the aged GO with cross-sectional height profiles; and DLS measurement of the aged GO (PDF)
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AUTHOR INFORMATION
Corresponding Author
were prepared on the same day, after 1 week of room temperature storage, and after 2 months. The first and last were further characterized by the techniques described below and also used for DNA-binding studies. Material Characterization. A field-emission scanning electron microscope (TESCAN MAIA 3) coupled with an energy dispersive spectrometer (Oxford Instruments, UK) was used to assess the morphology and elemental mapping of the obtained GO powder and drop-casted water dispersions. XPS was used to assess the chemical composition of the material. Wide-scan XPS surveys of all elements were performed, with subsequent high-resolution scans of the C 1s and O 1s regions. High-resolution XPS was performed using a ESCA Probe P
*E-mail:
[email protected] (M.P.). ORCID
Amir Masoud Pourrahimi: 0000-0001-5867-0531 Zdeněk Sofer: 0000-0002-1391-4448 Martin Pumera: 0000-0001-5846-2951 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the project Advanced Functional Nanorobots (reg. no. CZ.02.1.01/0.0/0.0/15_003/0000444 1615
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financed by the EFRR). C.L.M.P. acknowledges the financial support of the European Union’s Horizon 2020 Research and innovation programme under the Marie Skłodowska-Curie Actions IF grant agreement no. 795347. Z.S. was supported by the Czech Science Foundation (GACR No. 16-05167S) and by the specific university research (MSMT no. 20-SVV/2018). This work was created with the financial support of the Neuron Foundation for science support.
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