Direct Quantification of DNA Base Composition by Surface-Enhanced

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Direct Quantification of DNA Base Composition by Surface-enhanced Raman Scattering Spectroscopy Judit Morla-Folch, Ramon A. Alvarez-Puebla, and Luca Guerrini J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b01424 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 25, 2016

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The Journal of Physical Chemistry Letters

Direct Quantification of DNA Base Composition by Surface-enhanced Raman Scattering Spectroscopy Judit Morla-Folch,a,b Ramon A. Alvarez-Pueblaa,b,c* and Luca Guerrini.a,b*

a

Medcom Advance, Viladecans Business Park, Edificio Brasil, Bertran i Musitu 83-85, 08840

Viladecans, Barcelona, Spain. b

Centro Tecnológico de la Química de Catalunya and Universitat Rovira I Virgili, Carrer de

Marcel•lí Domingo s/n, 43007 Tarragona, Spain. c

ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Spain.

AUTHOR INFORMATION Corresponding Author * Email: [email protected] * Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Design of ultrasensitive DNA sensors based on the unique physical properties of plasmonic nanostructures has become one of the most exciting area in nanomedicine. However, despite the vast number of proposed applications, the determination of the base composition in nucleic acids, a fundamental parameter in genomic analyses and taxonomic classification, is still restricted to time-consuming and poorly sensitive conventional methods. Herein, we demonstrate the possibility of determining the base composition in single and double stranded DNA by using a simple, low cost, high throughput and label-free surface-enhanced Raman scattering (SERS) method in combination with cationic nanoparticles.

TOC GRAPHICS

KEYWORDS. DNA, plasmon, surface-enhanced Raman scattering, nanoparticles, taxonomic


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The characterization of nucleic acids (NAs) has become a major goal in fields such as medicine, genetics, forensics and drug screening.1-2 Plasmonic nanomaterials have been extensively incorporated into devices for the exploration of NAs by exploiting the unique optical properties generated by localized surface plasmon resonances (LSPRs).3-8 Due to the usually low amount of target DNA available, the development of optical strategies that could detect the genetic material with extremely high sensitivity (i.e.; PCR-free methods) represents a key requirement for their effective implementation into rapid, easy-to-use, cost-effective and high accuracy DNA sensors.5 In this regard, when plasmonic nanostructures such as gold and silver nanoparticles are employed to amplify the Raman scattering response of molecules located at the metallic surface (i.e.; surface-enhanced Raman scattering, SERS), sensitivity down to singlemolecule can be achieved.9 Such extraordinary sensitivity in combination with the rich structural information provided by Raman spectra turned SERS into a powerful bioanalytical tool.3-5, 7 The design of SERS biosensors for DNA detection largely involved indirect approaches incorporating secondary detection labels and bio-recognition elements (i.e.; oligonucleotide strands complementary to the target DNA).3-5, 7 However, extensive efforts have also been recently devoted to develop highly sensitive and reliable direct label-free SERS methods.10-14 DNA base composition is a fundamental parameter in genomic analyses15 and taxonomic classification.16 Traditional methods for screening of nucleobase quantification, such as HPLC, melting point, and buoyant density, are time-consuming, labor-intensive and normally requires relatively large amounts of DNA.17 Surprisingly, in the multiple SERS-based sensing approaches for DNA analysis reported in the literature, quantification of base composition in nucleic acids have been limited so far to synthetic short (12-mer) bi-polymeric Adenine/Cytosine sequences.11,


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Herein, we demonstrate the potential of SERS in the quantification of the base composition in

complex single- and double-stranded DNA (short and genomic). Cationic spermine-coated silver nanoparticles (AgNP@Sp) with a size of ca. 25 nm and a LSPR band centered at ca. 392 nm (Fig. 1A and Fig. S1), were prepared by simultaneous reduction of silver ions in the presence of spermine.12 The addition of DNA, either single strands or duplexes, induces a fast nanoparticle aggregation into stable clusters mediated by the electrostatic interaction between the negatively charged phosphate backbones and the positive metallic surface (Fig. 1B).12 DNA molecules act as an ‘electrostatic glue’ for AgNP@Sp, resulting in the entrapment of the nucleic acids at the interparticle junctions within the aggregates (i.e.; electromagnetic ‘hot spots’). As a result, illumination of the colloidal suspension with a 532 nm laser yields highly intense and reproducible averaged SERS spectra of DNA at nanograms per mL level (Fig. 1C),12, 19 while integration with microfluidic devices reduces the required amount of sample to picogram levels.13 Within these spectra, we can identify four broad spectral regions of interest: 500-820 cm-1, 820-1150 cm-1, 1150-1600 cm-1, and 1600-1750 cm-1 (Fig. 1C). In the first one, ring stretching bands of purine and pyrimidine residues dominate the spectral profile. The second spectral range mainly contains bands that originate from the deoxyribose-linked phosphodiester network, such as the intense band at ca. 1089 cm-1, ascribed to the symmetric stretching of the phosphodioxy moiety, νPO2-. Mostly in-plane ring vibrations of the nucleobases form a complex pattern of many overlapping features in the 1150-1600 cm-1 range, while the superimposition of carbonyl stretching modes yield a broad band approximately centered at 1640-1655 cm-1.12, 20-21


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Figure 1. (A) Extinction spectrum of positively-charged spermine-coated silver colloids (AgNP@Sp) and a representative TEM image of as-synthesized nanoparticles dried on a copper grid (scale bar equals to 20 nm). (B) Schematic outline of the DNA-driven aggregation of cationic nanoparticles into stable clusters in suspension, yielding (C) intense SERS spectra (21-bp double stranded DNA, ds1). The four broad spectral regions of interest are highlighted in different colours.

While structural classification of large biomolecules requires multivariate analysis of the Raman spectra, an univariate approach is more suited when relative quantifications are required.22 Identification of an internal standard is also necessary to remove the fluctuations of the absolute intensity measurements. In our case, the intense νPO2- band at ca. 1089 cm-1 was designated as the internal standard since it is poorly affected by DNA structural changes.11-12, 23 Additionally, the ratio between the overall content of phosphate groups and nucleobase units is constant and independent of the base composition. As spectral markers of the relative nucleobase content, we selected the relatively well-separated and intense ring modes in the lower spectral range. The relative intensities of these features display some degree of fluctuations within the first ca. 60 min upon the addition of DNA to the colloids (Fig. S2). For this reason, samples were incubated for 3 hours prior to the SERS analysis. Eleven 21-mer single stranded DNA (Table S1) and 21-base pair duplexes (Table S2) of different base sequence and composition were selected as probe samples to train the sensing system and extract the quantification parameters. Figure 2 and 3 show the SERS spectra of the


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probe samples in the sub 1150 cm-1 spectral range and their corresponding nucleobase content (in percentage). The spectra were normalized to the internal spectral standard at 1089 cm-1.

C+T A

C 600

G

700

800

Single strands ss1 ss2 ss3 ss4 ss5 ss6 ss7 ss8 ss9 ss10 ss11

900

1000

Raman shift (cm-1)

PO-2

1100

ss1 ss2 ss3 ss4 ss5 ss6 ss7 ss8 ss9 ss10 ss11

%A

%T

%C

%G

28.6 19.0 33.3 19.1 38.1 9.5 23.8 38.1 47.6 33.4 33.3

19.0 28.6 23.8 33.3 19.0 19.0 38.1 23.8 33.4 47.6 33.3

23.8 28.6 23.8 38,1 23.8 42.9 14.3 23.8 9.5 9.5 0.0

28.6 23.8 19.1 9.5 19.0 28.6 23.8 14.3 9.5 9.5 33.3

Figure 2. (A) SERS spectra of 21-mer single stranded DNA sequences of different composition and base sequence, in the 580-1160 cm-1 spectral region. All spectra were normalized to the νPO2- band at ca. 1089 cm-1. The illustrated spectra are from averaging 8 independent repeats per sample.

Baseline correction was identically applied to all spectra to remove fluctuations of the background signal. A general vibrational assignment is also included in the figure. In the case of single strands (Fig. 2), we can clearly distinguish the intense ring breathing modes of guanine (G) at ca. 684 cm-1 and adenine (A) at ca. 733 cm-1, while the intense feature at ca. 792 cm-1 contains two distinct components: the cytosine (C) and the thymine (T) ring breathing bands centered at ca. 793 and 790 cm-1, respectively.12 An additional C band at ca. 599 cm-1 is ascribed to a ring bending mode.24 Differently, a significant reshaping of the overall spectral profile takes place when the individual strands hybridize in the double helix (Fig. 3). Among others, we highlight the notable shift of the nucleobase ring breathing modes of G, A and C+T to ca. 676,


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729 and 784 cm-1, respectively (Fig. 3), while the C ring bending vibration undergoes a significant intensity drop and a G ring deformation strongly emerges at ca. 620 cm-1.12

Duplexes

A ds1 ds2 ds3 ds4 ds5 ds6 ds7 ds8 ds9 ds10 ds11

C+T

G

500

G G C 600

700

800

900

Raman shift (cm-1)

ds1 ds2 ds3 ds4 PO-2 ds5 ds6 ds7 ds8 ds9 ds10 1000 1100 ds11

%A

%T

%C

%G

23.8 26.2 26.2 35.7 30.9 19.1 19.1 40.5 0 50 26.2

23.8 26.2 26.2 35.7 30.9 19.1 19.1 40.5 0 50 23.8

26.2 23.8 23.8 14.3 19.1 30.9 30.9 9.5 50 0 21.4

26.2 23.8 23.8 14.3 19.1 30.9 30.9 9.5 50 0 28.6

Figure 3. SERS spectra of 21-bp double stranded DNA chains of different composition and base sequence, in the 450-1150 cm-1 spectral region. All spectra were normalized to the νPO2- band at ca. 1089 cm-1. The illustrated spectra are from averaging 8 independent repeats per sample.

Based on the spectral analysis of single strands, it is possible to select the peak heights of the strong and well-separated features at ca. 599, 684 and 733 cm-1 as the quantitative spectral markers for C, G and A relative content, respectively. On the other hand, no pure thymine features can be isolated in the SERS spectra. However, in principle it would be possible to extract the relative thymine content from the composed C+T ring breathing band at ca. 792 cm-1 once determined the initial C content via the analysis of the 599 cm-1 feature. Thus, through an iterative process based on the outcome of the SERS measurements of the single strand probes in Figure 2 (see Supp. Inf., pg. S5, for a detailed description), we progressively adjusted the relative contributions of the C and T residues to the peak height of the 792 cm-1 band to an optimal ratio of (aC + 0.22 × bT), where a and b are the relative content of C and T in the strand,


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respectively (i.e.; one cytosine contributes approximatively 4 times more than one thymine to the peak height of the 792 cm-1 band). This is consistent with previous normal Raman studies reported in the literature.25 However, in the SERS spectra of the duplexes the C band at ca. 599 cm-1 can no longer be selected as a reliable spectral marker due to its intrinsic weakness and partial overlapping with the emerging G feature at ca. 620 cm-1. Nonetheless, based on the Chargaff’s first parity rule of nucleotide base composition in double-stranded DNA (i.e.; [A] = [T] and [G] = [C]),26 it is still possible to estimate the relative GC and AT base content in DNA duplexes from the peak height ratios of G band at ca. 676 cm-1 and A band at ca. 729 cm-1 (normalized to the internal standard νPO2- band). Thus, from the SERS analysis of the 21-mer strands and 21-bp duplexes, we calculated the normalized peak height values for each spectral marker corresponding to 1% of (i) individual nucleobase content in single stranded DNA and (ii) combined AT or GC content in DNA duplexes (Table S3). The validity of such nucleobase quantification approach was verified by selecting three different DNA chains as unknown samples (Table S4). Specifically, we acquired (Fig. S3) the SERS spectra of a 21-mer single-stranded sequence (ssDNA21), a 21-bp synthetic duplex (dsDNA21) and genomic DNA from calf thymus (ctDNA). The relative quantifications of each individual nucleobase for ssDNA and AT/GC contents in duplexes, based on the peak height ratios acquired from their direct SERS analysis, are represented in Figure 4 together with the reference values. The results show an outstanding accuracy of the direct SERS method at predicting the relative nucleobase contents in DNA chains of different composition and length (from ca. 7 nm long 21-bp dsDNA to 10-15 kb DNA from calf thymus).


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Figure 4. Quantification of nucleobase content (%) in the 21-mer single strand ssDNA21 and quantification of CG and AT base content (%) in the 21-bp double stranded DNA (dsDNA21) and genomic DNA from calf thymus (ctDNA). Error bars equal to two standard deviations (N = 8).

In summary, we have developed a simple direct SERS method for the ultrasensitive, fast and label-free relative quantification of the nucleobase content in DNA. The peak heights of characteristic nucleobase markers were normalized to the internal standard (an intense phosphate-related feature which is poorly affected by DNA structural changes) to yield spectral ratios that can be quantitatively correlated with the relative number of purine/pyrimidine bases in the chain. Differently to indirect optical approaches based on DNA-nanoparticle conjugates, this


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simple, inexpensive and flexible method can straightforwardly be applied to DNA samples independently of their size (from short to long genomic DNA), conformation (single-stranded vs. duplex), and base sequence. EXPERIMENTAL METHODS Materials. All materials were obtained from Sigma-Aldrich. 21-mer single-stranded DNA were all purchased from Eurofins Genomics (Ebersberg, Germany). Deoxyribonucleic acid from calf thymus (Type XV, Activated, lyophilized powder) was purchased from Sigma Aldrich. Short dsDNA chains were prepared by annealing (90 °C for 10 min) equimolar mixtures of the corresponding complementary strands in PBS 0.3 M. Synthesis of positively-charged silver colloids. 20 µL of a AgNO3 0.5 M solution and 7 µL of a spermine tetrahydrochloride 0.1 M solution were subsequently mixed with 10 mL of Milli-Q water. After 20 min of stirring, 250 µL of NaBH4 (0.01 M) were rapidly injected into the mixture. Vials employed for particle preparation and storage were previously coated with polyethyleneimine (PEI) to prevent the adhesion of positively-charged nanoparticles to glass surfaces, as described elsewhere.12 Silver colloids were left to equilibrate overnight and the deposit that settled at the bottom of the vial was removed from the sample. Colloids are composed of quasi-spherical nanoparticle of ca. 25 nm diameter characterized by a narrow localize surface plasmon resonance (LSPR) centered at ca. 392 nm, and a ζ-potential of ca. +41 mV. The final nanoparticle concentration of AgSp (ca. 1.1 nM) was calculated according to the Lambert-Beer’s law by using an extinction coefficient of 54.8 × 108 M-1 cm-1 obtained from the literature.27 Sample preparation. Samples for SERS measurements were prepared by mixing 150 µL of AgNP@Sp (ca. 1.1 nM) with 12 µL of DNA solutions (10 µM for ssDNA; 5 µM for short


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dsDNA and 15 mg/mL for calf thymus DNA). Samples were left unperturbed for 3 hours, and then quickly sonicated before being investigated by SERS. Instrumentation. A Renishaw InVia Reflex confocal microscope equipped with a 532 nm laser was used to carry out the SERS analysis. The laser was focused on the colloidal suspension with a macrolens providing an efficient spot of 0.6 mm. This experimental set-up also avoids uncontrolled formation of aggregates via light-induced trapping.28 8 different replications per each sample were acquired under the same experimental conditions (10 s exposure time, 20 accumulations and ca. 34.5 mW laser power at the sample). UV-vis spectra were recorded using a Thermo Scientific Evolution 201 UV-visible spectrophotometer. TEM was performed with a JEOL JEM-1011 transmission electron microscope. ζ-potential measurements were performed on a Malvern Nano Zetasizer. ASSOCIATED CONTENT Supporting Information. Particle characterization, compositional tables for the studied nucleic acids, description of the iterative process for estimation of relative contributions of cytosine and thymine residues to the peak height of the 792 cm-1 band in ssDNA, influence of incubation time on the obtained SERS spectra, and additional SERS measurements. The following files are available free of charge. AUTHOR INFORMATION Notes The authors declare no competing financial interests. ACKNOWLEDGMENT


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The work was funded by Ministerio de Economia y Competitividad (CTQ2014-59808R), European Research Council (PrioSERS FP7/2014 623527), Generalitat de Catalunya (2014SGR-480), Agencia de Gestió d’Ajuts Universitaris de Recerca (AGAUR 2014 DI 054), Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya, and Medcom Advance SA. REFERENCES (1) Kayser, M.; de Knijff, P. Improving Human Forensics through Advances in Genetics, Genomics and Molecular Biology. Nat. Rev. Genet. 2011, 12, 179-192. (2) Pinheiro, A. V.; Han, D. R.; Shih, W. M.; Yan, H. Challenges and Opportunities for Structural DNA Nanotechnology. Nat. Nanotechnol. 2011, 6, 763-772. (3) Peng, H. I.; Miller, B. L. Recent Advancements in Optical DNA Biosensors: Exploiting the Plasmonic Effects of Metal Nanoparticles. Analyst 2011, 136, 436-447. (4) Fong, K. E.; Yung, L. Y. L. Localized Surface Plasmon Resonance: A Unique Property of Plasmonic Nanoparticles for Nucleic Acid Detection. Nanoscale 2013, 5, 12043-12071. (5) Vo-Dinh, T.; Fales, A. M.; Griffin, G. D.; Khoury, C. G.; Liu, Y.; Ngo, H.; Norton, S. J.; Register, J. K.; Wang, H. N.; Yuan, H. Plasmonic Nanoprobes: From Chemical Sensing to Medical Diagnostics and Therapy. Nanoscale 2013, 5, 10127-10140. (6) Giannini, V.; Fernandez-Dominguez, A. I.; Heck, S. C.; Maier, S. A. Plasmonic Nanoantennas: Fundamentals and Their Use in Controlling the Radiative Properties of Nanoemitters. Chem. Rev. 2011, 111, 3888-3912. (7) Alvarez-Puebla, R. A.; Liz-Marzan, L. M. SERS-Based Diagnosis and Biodetection. Small 2010, 6, 604-610.


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(16) Fournier, P. E.; Suhre, K.; Fournous, G.; Raoult, D. Estimation of Prokaryote Genomic DNA G+C Content by Sequencing Universally Conserved Genes. Int. J. Syst. Evol. Microbiol. 2006, 56, 1025-1029. (17) Hua, N.-P.; Naganuma, T. Application of Ce for Determination of DNA Base Composition. Electrophoresis 2007, 28, 366-372. (18) Papadopoulou, E.; Bell, S. E. J. Label-Free Detection of Nanomolar Unmodified Singleand Double-Stranded DNA by Using Surface-Enhanced Raman Spectroscopy on Ag and Au Colloids. Chem.-Eur. J. 2012, 18, 5394-5400. (19) Masetti, M.; Xie, H.-n.; Krpetić, Ž.; Recanatini, M.; Alvarez-Puebla, R. A.; Guerrini, L. Revealing DNA Interactions with Exogenous Agents by Surface-Enhanced Raman Scattering. J. Am. Chem. Soc. 2015, 137, 469-476. (20) Benevides, J. M.; Overman, S. A.; Thomas, G. J. Raman, Polarized Raman and Ultraviolet Resonance Raman Spectroscopy of Nucleic Acids and Their Complexes. J. Raman Spectrosc. 2005, 36, 279-299. (21) Turpin, P. Y.; Chinsky, L.; Laigle, A.; Jolles, B. DNA-Structure Studies by Resonance Raman-Spectroscopy. J. Mol. Struct. 1989, 214, 43-70. (22) Kumar, S.; Verma, T.; Mukherjee, R.; Ariese, F.; Somasundaram, K.; Umapathy, S. Raman and Infra-Red Microspectroscopy: Towards Quantitative Evaluation for Clinical Research by Ratiometric Analysis. Chem. Soc. Rev. 2016, 45, 1879-1900. (23) Duguid, J. G.; Bloomfield, V. A.; Benevides, J. M.; Thomas, G. J. DNA Melting Investigated by Differential Scanning Calorimetry and Raman Spectroscopy. Biophys. J. 1996, 71, 3350-3360.


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Direct Quantification of DNA Base Composition by Surface-Enhanced

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