ARTICLE pubs.acs.org/ac
Direct Detection of Point Mutations in Nonamplified Human Genomic DNA Roberta D’Agata,† Giulia Breveglieri,‡ Laura M. Zanoli,§ Monica Borgatti,‡ Giuseppe Spoto,*,†,|| and Roberto Gambari*,‡,^ †
Dipartimento di Scienze Chimiche, Universita di Catania, Viale Andrea Doria 6, I-95125 Catania, Italy Centro di Biotecnologie, Universita di Ferrara, via Fossato di Mortara 64b, 44121 Ferrara, Italy § Scuola Superiore di Catania, c/o Dipartimento di Scienze Chimiche, Universita di Catania, Viale Andrea Doria 6, Catania, Italy Istituto Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, Catania, Italy ^ Dipartimento di Biochimica e Biologia Molecolare, Universita di Ferrara, via Fossato di Mortara 74, 44121 Ferrara, Italy
)
‡
bS Supporting Information ABSTRACT: Ultrasensitive detection protocols not requiring polymerase chain reaction (PCR)-mediated target DNA amplification are expected to significantly improve our possibilities in several research and diagnostic applications for which minute cell quantities are available. For this reason we have tested a nanoparticle-enhanced surface plasmon resonance imaging (SPRI) sensing strategy to detect point mutations in nonamplified genomic DNA. We have used genomic DNAs, not subject to costly, time-consuming, and prone to contamination PCR-based amplification procedures, obtained from both healthy individuals and homozygous or heterozygous patients affected by β-thalassemia, in order to demonstrate the specificity and the sensitivity of the described sensing strategy. The assay we describe is ultrasensitive and convenient. Attomolar concentrations of target genomic DNA are detected, DNAs from healthy individuals and homozygous or heterozygous patients affected by β-thalassemia are discriminated, and only simple manipulations of the genetic samples are required before the analysis. The proposed ultrasensitive detection of DNA point mutations involved in genomic disorders possibly represents an important advantage in several biomedical applications.
S
ingle-nucleotide polymorphisms (SNPs) represent the most abundant class of alterations in the human genome,1 and their identification has important applications in molecular diagnostics and genetic risk assessment.2,3 Therefore newer, faster, and more efficient techniques for SNPs detection are of wide interest. SNPs are mostly detected by using platforms exploiting allele-specific hybridization, operating in an array format4 or using real-time polymerase chain reaction (PCR).5 Sequence variations are detected after the amplification of the genomic DNA because of both the reduced amount of genomic material available as well as the limited sensitivity of the detection protocol. Thus far, DNA detection has relied on amplification procedures based largely on PCR, even though they are costly, difficult to parallelize, and prone to generate severe DNA sample contaminations.6 We propose in this paper a procedure to detect SNPs in attomolar concentrations of target genomic DNA, not requiring PCRmediated target DNA amplification Efforts have been recently made in identifying innovative and ultrasensitive DNA detection protocols which can be performed without PCR.79 Most of them exploit strategies for signal amplification based on the use of enzymes10,11 or metallic nanostructures.12 In particular, peculiar chemical and physical properties of gold nanoparticles (AuNPs) have been used to obtain an ultrasensitive DNA detection.13 Recently, we have shown that r 2011 American Chemical Society
ultrasensitive detection of nonamplified genetically modified organism (GMO) soybean genomic DNA can be obtained by using a nanoparticle-enhanced surface-plasmon resonance imaging (SPRI) platform and peptide nucleic acid (PNA) probes.14,15 PNAs are DNA mimics in which the negatively phosphate deoxyribose backbone is replaced by a neutral N-(2-aminoethyl)glycine linkage. They have been shown to be able to improve both selectivity and sensitivity in targeting complementary DNA and RNA sequences.16 PNAs have been proposed as valuable alternatives to oligonucleotide probes useful to build neutral surfaces.17 SPRI is a label-free technique able to detect molecular and biomolecular interactions occurring on a metallic surface,1820 differing from the standard spectral SPR because imaging capability enables analyses to be carried out with high throughput and low sample consumption by coupling microfluidic devices with the SPRI apparatus.21 It represents a versatile platform for both nucleic acids and proteins detection offering the possibility to operate with complex matrixes.22 In this work we describe the ultrasensitive nanoparticleenhanced SPRI detection of SNPs in nonamplified human Received: August 19, 2011 Accepted: October 7, 2011 Published: October 07, 2011 8711
dx.doi.org/10.1021/ac2021932 | Anal. Chem. 2011, 83, 8711–8717
Analytical Chemistry
Figure 1. Scheme of the human β-globin gene is shown with the sequence targeted by PNA-N and PNA-M probes. The position of the β39 C>T point mutation is also shown. The sequence of DNAβ39 11mer biotinylated oligonucleotide immobilized on gold nanoparticles is also shown with the complementary target sequence.
genomic DNAs carrying the mutated β39-globin gene sequence. The mutated sequence is involved in a severe type of β-thalassemia, a group of hereditary blood disorders causing anomalies in the synthesis of the β chains of adult hemoglobin A (α2ββ2) and generating in some cases severe anemia. Currently, molecular diagnosis of β-thalassemia is carried out by using different methodologies, most of which are based on PCR.23,24 Only recently, whole genome sequencing of the maternal plasma DNA consisting of both maternal-derived DNA as well as fetalderived DNA has been shown to enable the detection of the fetal β-thalassemia alleles against the background of the maternal DNA.25 In the context of molecular diagnosis, biosensor technologies are expected to significantly improve the possibilities for fast, simple, and reliable diagnostics of genetic diseases, including β-thalassemia.2629
’ EXPERIMENTAL SECTION Materials and Reagents. Wild-type streptavidin (WT-SA) was purchased from Invitrogen (Italy). Mixed cellulose ester membrane filters were purchased from Whatman (U.K.). Trisodium citrate dihydrate, tetrachloroauric(III) acid, ethanol, dimethyl sulfoxide (DMSO), hexane, sodium hydroxide solutions (10 M in water), and dithiobis(N)succinimidylpropionate (DTSP) were purchased from Sigma-Aldrich (Italy). Phosphatebuffered saline (PBS) solutions (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) were obtained from Amresco (Italy). Biotinylated oligonucleotide DNAβ39 (50 -AGCAGCCTAAG-30 ) (Figure 1) was purchased from Thermo Fisher Scientific, Inc. PNA probes (Figure 1) (PNA-N, 50 -(AEEEA)2-CTCTGGGTCCAA-30 and PNA-M, 50 -(AEEEA)2-CTCTAGGTCCAA-30 ) were purchased from Panagene Inc. Ultrapure water (Milli-Q Element, Millipore) was used for all the experiments. Extraction, Purification, and Characterization of Genomic DNA Samples. Blood samples were collected using test tubes containing a standard anticoagulant. The isolation of blood samples from four healthy individuals (normal) and 14 β-thalassemia patients (seven heterozygous and seven homozygous for the β39 mutation) was approved by the Ethical Committee of S. Anna Hospital (Ferrara). Informed consent was obtained, and the experiments were conducted in agreement with the Declaration of Helsinki.
ARTICLE
Genomic DNA was isolated from leucocytes in peripheral blood of normal subjects and β-thalassemia patients by using the Qiagen blood and cell culture DNA mini kit according to manufacturer’s instructions. The DNA extraction method was chosen in order to get contaminant-free genomic DNA suitable for all sensitive downstream applications. The method takes advantage of the multiplex purification of DNAs extracted from less than 1 mL of blood with no need for expensive equipments and toxic or hazardous reagents. Purified DNA samples were analyzed by gel electrophoresis (Supporting Information Figure S11) and quantified by UV spectrophotometric measurement (1 OD260 corresponding to a dsDNA concentration of 50 ng μL1). Patients with classic β-thalassemia trait (Supporting Information Table S1) were identified by genotyping their β-globin gene by PCR amplification and DNA sequencing. PCR amplification was performed by using β-globinF (forward, 50 -GTGCCAGAAGAGCCAAGGACAGG-30 ) and β-globinR (reverse, 50 -AGTTCTCAGGATCCACGTGCA-30 ) primers. The primers were designed in order to amplify a 641 bp region of the human β-globin gene containing sequences carrying the most frequent β-thalassemia point mutations in Italy. An amount of 300 ng of genomic DNA were PCR amplified by using DreamTaq DNA polymerase (Fermentas). Each PCR amplification was performed in a solution (100 μL) containing 1 DreamTaq buffer (KCl, (NH4)2SO4, 20 mM MgCl2), 33 μM dNTPs, 150 ng of PCR primers, and 1.25 U of DreamTaq DNA polymerase. The 35 amplification cycles used were as follows: denaturation, 95 C, 30 s; annealing, 65 C, 20 s; elongation, 72 C, 1 min. PCR products were analyzed by agarose gel electrophoresis (Supporting Information Figure S12). β-GlobinF β-globinR PCR products were purified with MicroCLEAN (Microzone Limited) and sequenced by using the ABI PRISM BigDye terminator cycle sequencing ready reaction kit, v 2.0 (Applied Biosystems) in order to identify the patients’ genotypes. With the aim to investigate the whole β-globin gene, different sequencing primers, recognizing different sequence positions, were employed. Sequence reactions were performed in a final volume of 20 μL, containing 60 ng of PCR template, 3.2 pmol of sequencing primer, 1 sequencing buffer and 8 μL of terminator ready reaction mix. A total of 45 amplification cycles were performed, as follows: denaturation, 96 C, 10 s; annealing, 65 C, 5 s; elongation, 65 C, 3 min. A denaturing 4% polyacrylamide gel electrophoresis was then carried out in an automated ABI PRISM 377 DNA sequencer (Applied Biosystems), and final sequence data were analyzed by Sequencing Analysis 3.3 (Applied Biosystems) and Chromas Lite 2.01 (Copyright 20032008 Technelysium Pty Ltd.) softwares (Supporting Information Figure S13). Synthesis and Functionalization of Gold Nanoparticles. Gold nanoparticles (AuNPs) were synthesized by citrate reduction of HAuCl4 3 3H2O according to methods elsewhere described.30 Briefly, 20 mL of trisodium citrate (38.8 mM) was quickly added with vigorous stirring to 200 mL of a boiling solution of HAuCl4 3 3H2O (1 mM). The color of the solution changed from pale yellow to deep red in a few seconds. A complete reduction of trisodium citrate was obtained after 68 min under boiling. The solution was cooled to the room temperature and filtered through a 0.45 μm mixed cellulose ester membrane filter. The AuNPs solution was stored at 4 C in a clean brown glass bottle. Similar conditions assured the nanoparticles stability for several months. AuNPs were characterized by UVvis spectroscopy (Nanodrop 1000 and Agilent 8453 spectrophotometers) and by transmission electron microscopy 8712
dx.doi.org/10.1021/ac2021932 |Anal. Chem. 2011, 83, 8711–8717
Analytical Chemistry
ARTICLE
(TEM) (Jeol JEM-2000 FX II, operating at 200 kV). The mean diameter of AuNPs was 20 ( 5 nm (Supporting Information Figure S2c). AuNPs were conjugated to the 30 end of biotinylated DNAβ39 with an 11-mer sequence (50 -AGCAGCCTAAG-30 , Tm = 34.0 C) as follows: 500 μL of a solution obtained by diluting (1:1) AuNPs with H2O was added to 10 μL of a 1 mg mL1 streptavidin solution after adjusting the pH to 8.5 (NaOH 0.1 M). The solution was incubated on ice for 1 h and centrifuged (12 500 rpm, 30 min, 23 C). After the separation of the liquid phase, the modified AuNPs were suspended with 90 μL of water. An amount of 10 μL of DNAβ39 (100 μM in H2O) was added to the streptavidinconjugated AuNPs suspension, and the mixture was incubated for 30 min. After the centrifugation and the removal of the supernatant solution DNAβ39-conjugated AuNPs were suspended in PBS buffer. The actual concentration of DNAβ39-conjugated AuNPs stock solutions was obtained from UVvis spectroscopy (ε528 = 2 108 M1 cm1) (Supporting Information Figure S2, parts a and b).31 Conjugated AuNPs stock solutions suitable for the SPRI ultrasensitive detection were characterized by λmax = 528 ( 1 nm and were between 10 and 30 nM in concentration. Moreover, the A260/A528 ratio was between 1 and 1.2. Conjugated AuNPs for SPRI signal enhancement were obtained by diluting DNAβ39-conjugated AuNPs stock solutions. The selection of an appropriate DNAβ39-conjugated AuNPs final concentration is critical for the success of the experiments. The described experiments were conducted by using 0.1 nM solutions (in PBS). SPRI Experiments. All SPRI experiments were carried out by using an SPR imager apparatus (GWC Technologies, U.S.A.). SPR images were analyzed by using the V++ software (version 4.0, Digital Optics Limited, New Zealand) and the ImageJ 1.32j software package (National Institutes of Health, U.S.A.). SPRI provides data as pixel intensity units (0255 scale). Data were converted into percentage of reflectivity (%R), or Δ%R in the case of difference images, by using the formula %R ¼ 100 0:85Ip =Is
ð1Þ
where Ip and Is refer to the reflected light intensity detected using p- and s-polarized light, respectively. Experiments were carried out by sequentially acquiring 15 frames averaged SPR images with 5 s time delay between them. Kinetic data were obtained by plotting the difference in percent reflectivity (Δ%R) from selected regions of interest (ROIs) of SPR images as a function of time. The selected ROIs were chosen in order to include all the SPR chip area involved by the surface interaction experiment. All the SPRI experiments were carried out at room temperature. A microfluidic device with six parallel microchannels (80 μm depth, 1.4 cm length, 400 μm width) and circular reservoirs (diameter = 400 μm) at the ends of each channel was used for the study, in order to allow an independent control of interactions occurring on six different regions of the SPRI gold chip surface. The device was fabricated in poly(dimethylsiloxane) (PDMS) polymer through the replica molding technique.32 PEEK tubes (UpChurch Scientific) were inserted in the circular reservoirs in order to connect the PDMS microfluidic cell to an Ismatec IPC (Ismatec SA, Switzerland) peristaltic pump. The microfluidic device was assembled by fixing the PDMS mold on the SPRI gold chip surface (Supporting Information Figure S10). A refractive index matching liquid was used to obtain the optical contact between the gold chip and the prism.
A strict cleaning procedure of the fluidic system was adopted in order to minimize contaminations and memory effects. The fluidic system was washed with ultraclean water (37 C, for 2 h) after each experiment and with PBS buffer for at least 24 h before each experiment. Every 3 weeks the system was cleaned as follow: 0.5% sodium dodecyl sulfate (SDS) (10 min), 6 M urea (10 min), 1% acetic acid (10 min), 0.2 M NaHCO3 (10 min), ultraclean water 37 C (30 min), PBS buffer (at least 24 h). Cleaning procedures were coupled to both a random exchange of tubing connecting the microfluidic device to the peristaltic pump as well as a change of PNA-N and PNA-M probes immobilization sequence. Moreover, in order to avoid artifacts generated by contaminations of the PDMS microfluidic device, normal, heterozygous, and homozygous DNA samples were adsorbed on PNA-N- and PNA-M-modified surfaces by loading each of them into neighboring microchannels which were different from experiment to experiment. The use of further optimized cleaning procedures and/or disposable fluidic devices are expected to help shorten cleaning procedure duration. Gold chips (GWC Technologies, U.S.A.), previously functionalized with DTSP, were used to immobilize PNA-N and PNA-M probes. The procedure adopted for gold chip functionalization33 was the following: bare gold chip were immersed in a DTSP solution (4 mM in DMSO) for 48 h. Modified chips were then thoroughly rinsed with DMSO, ethanol, and ultrapure water, respectively. PNA-N and PNA-M probes were immobilized on DTSP-modified gold chips through the amine-coupling reaction between N-hydroxysuccinimidyl (NHS) ester ends of DTSP and the N-terminal group present at 50 -position of AEEEA linker. The spatially separated immobilization of PNA-N and PNA-M probes was obtained by injecting PNA-N or PNA-M solutions (0.1 μM in PBS, flow rate 5 μL min1) (Supporting Information Figure S1) into parallel microchannels in contact with the DTSP-modified gold surface. AEEEA spacers were used in order to minimize surface effects caused by the steric hindrance of immobilized systems. A mean thickness of 1.6 nm was calculated for the deposited probe layer.34,35 A mean PNA-N and PNA-M surface coverage of about 3 1012 molecules cm2 was calculated by multiplying the mean adlayer thickness value by the bulk number density of the adsorbate (molecules nm3).36 Before SPRI analyses genomic DNA samples were fragmented by sonication (3 min, ELMA Transsonic T480/H-2) and vortexing (1 min, IKA Vortex GENIUS 3) and denatured by heating at 95 C for 5 min. Genomic DNAs randomly sheared by sonication generated fragments between 300 and 2000 bp. It has been reported that fragments of approximately 400 bp are of sufficient size to localize the fragment within the human genome with a high degree of statistical certainty.37 Strands reassociation was prevented by cooling on ice the samples (1 min) before their introduction into the SPRI microfluidics apparatus. DNA solutions were used no more than 1 min after the previous treatment. SPRI genomic DNA hybridization experiments were performed at room temperature by using 300 μL of 5 pg μL1 (∼2.6 aM) fragmented and denatured genomic DNA solutions which were adsorbed (flow rate 10 μL min1) on PNA-N- and PNA-Mfunctionalized surfaces. The two different PNA probes were useful both to discriminate between normal, homozygous, and heterozygous DNAs as well as to avoid the use of external controls which were difficult to be obtained for this specific application. In fact, since biological samples from humans and not certified materials were used in this work, differences in the 8713
dx.doi.org/10.1021/ac2021932 |Anal. Chem. 2011, 83, 8711–8717
Analytical Chemistry
ARTICLE
Figure 2. Pictorial description of the nanoparticle-enhanced SPRI strategy used to detect the normal βN/βN, heterozygous β39/βN, and homozygous β39/β39 genomic DNAs. In order to simplify the pictorial representation only specifically adsorbed DNA is shown. Nonspecifically adsorbed DNA is also present on the surface and contributes to generate the SPRI-detected signal. PNA-N and PNA-M specifically recognize the normal β-globin and the mutated β39-globin genomic sequences, respectively.
final concentration of the analyzed samples would affect the level of the SPRI responses by preventing a reliable comparison between the signals generated by the different DNA samples. Solutions of 5 pg μL1 DNA were prepared no more than 48 h before the experiments from 100 pg μL1 solutions obtained by diluting DNA stock solutions (Supporting Information Table S1). The direct adsorption of DNA solutions (βN/βN, β39/N, β39/β39) on PNA-N- and PNA-M-functionalized surfaces did not generate SPRI responses useful for samples discrimination. Supporting Information Figure S3 shows representative Δ%R over time obtained for the absorption of the above-mentioned 5 pg μL1 solutions on PNA-N- and PNA-M-functionalized surfaces.
’ RESULTS AND DISCUSSION The method we describe here allowed us to discriminate between normal, β39 homozygous, and β39 heterozygous nonamplified genomic DNAs down to a 2.6 aM (2.6 1018 M) concentration. Figure 1 shows the sequences of PNA-N and PNA-M probes, specific for the normal β-globin sequence and the β39 mutated sequence, respectively, as well as the biotinylated DNAβ39 11mer oligonucleotide employed for the functionalization of gold nanoparticles (AuNPs) and targeting molecular hybrids constituted by PNA probes and genomic DNAs. The strategy used for the ultrasensitive detection of SNPs in nonamplified genomic DNA is shown in Figure 2. The genomic DNA was extracted from blood samples of healthy individuals and β-thalassemia patients, quantified and diluted to 5 pg μL1. Typical experiments were performed by detecting SPRI multiplex responses from three genotypically different DNA samples: normal (βN/βN), homozygous (β39/β39), and heterozygous
(β39/βN) for the β39 point mutation. Before the SPRI detection, DNA samples were sonicated, denatured by heating at 95 C for 5 min, rapidly cooled, and incubated for 1 min on ice immediately before the SPRI analysis. An amount of 300 μL of each sample was then directly fluxed into each of six microchannels of the SPRI fluidic system (Supporting Information Figure S10) in order to allow the direct interaction of each of the three samples with PNA-N- and PNAM-functionalized surfaces (Figure 2, parts a and b). The specific SPRI response patterns obtained when normal, homozygous, or heterozygous DNAs were each allowed to interact with the two different PNA probes provided a robust experiment control. In fact, whereas normal DNA (βN/βN) was expected to interact with only the PNA-N probe (Figure 2c), different interactions were expected from homozygous and heterozygous DNAs, i.e., interaction between homozygous DNA (β39/β39) and only the PNA-M probe and interactions between heterozygous DNA (β39/βN) and both PNA-N and PNA-M probes. The direct absorption of the above-mentioned 5 pg μL1 solutions of shared genomic DNAs on the PNA-N- and PNA-M-functionalized surfaces generated SPRI signals close to that of instrumental noise (Supporting Information Figure S3). The observed signal trend is in line with previous observations15 and with the SPRI response to be expected when a very low surface coverage of bound DNA target (lower than 3 1012 molecules cm2 in the discussed case) is obtained. Ultrasensitive detection of the genomic DNA was achieved by using AuNPs conjugated to the DNAβ39 11-mer oligonucleotide, complementary to an exposed tract of the target DNA not involved in the hybridization with the PNA probe (Figures 1 and 2d). After the capture of unamplified genomic DNAs carrying the normal or the β39 mutated gene sequences by the complementary 8714
dx.doi.org/10.1021/ac2021932 |Anal. Chem. 2011, 83, 8711–8717
Analytical Chemistry
ARTICLE
Figure 3. Time-dependent SPRI curves obtained after the adsorption of conjugated AuNPs on normal βN/βN (a, sample 83), heterozygous β39/βN (b, sample 5), and homozygous β39/β39 (c, sample 6) DNAs previously adsorbed to the surface immobilized PNA-N (red curve) and PNA-M (blue curve) probes. Solutions of 5 pg μL1 genomic DNAs were used for the experiments. A representative SPR difference image (d) demonstrating the DNA parallel detection is also shown.
PNA probe(s), the conjugated AuNPs were fluxed into the microchannels in order to enhance detectability. By using this approach, a significant increase of the detected SPRI signals was obtained (Figure 2, parts e and f). Figure 3 shows the SPRI change in percent reflectivity (Δ%R) over time (Figure 3ac) and a representative SPR difference image (Figure 3d) obtained after the nanoparticleenhanced SPRI detection of nonamplified normal (Figure 3a), heterozygous (Figure 3b), and homozygous (Figure 3c) genomic DNAs (Supporting Information Figures S4S9). The detected SPRI responses were in line with those expected. In fact, normal (βN/βN) DNA preferentially hybridizes with the PNA-N probe, whereas homozygous (β39/β39) DNA preferentially hybridizes with the PNA-M probe. Heterozygous sample hybridizes with both PNA-N and PNA-M probes with strikingly similar efficiency. Figure 4 shows average results from replicate experiments carried out by using genomic DNAs from four normal individuals (βN/βN) and 14 β-thalassemia patients (seven homozygous β39/β39 and seven heterozygous β39/βN) (Supporting Information Table S1). In particular, Figure 4 shows the ratio between the SPRI responses (Δ%R) referred to the PNA-N
probe (Δ%RPNA‑N) and the PNA-M probe (Δ%RPNA‑M) when the same DNA target was detected. Homozygous (β39/β39), heterozygous (β39/βN), and normal (βN/ βN) DNAs generated Δ%RPNA‑N and Δ%RPNA‑M responses whose ratios were different from one another (two-tailed t test, level 99%, P < 0.0001). Time-dependent SPRI response shapes (Figure 3ac and Supporting Information Figures S4S9) were generated by the functionalized AuNPs mass transport limitation of the binding interactions. The level of the time-dependent SPRI responses was significantly affected by tiny variations in the final concentration of conjugated AuNPs solutions. In fact, relative standard deviation (sr) values ranging from 0.75 to 0.90 were calculated for each group of replicated experiments. However, significantly lower sr values were obtained when the ratio between the SPRI responses referred to the PNA-N probe (Δ%RPNA‑N) and the PNA-M probe (Δ%RPNA‑M) (Figure 4) were considered (homozygous sr = 0.32, heterozygous s r = 0.05, normal sr = 0.15). By assuming a molecular weight of about 1.9 1012 for the human genome (genome size about 2.9 Gb),38 we conclude that 8715
dx.doi.org/10.1021/ac2021932 |Anal. Chem. 2011, 83, 8711–8717
Analytical Chemistry
Figure 4. Mean Δ%RPNA‑N/Δ%RPNA‑M ratio values obtained from replicated experiments aimed at detecting 5 pg μL1 solutions of normal (βN/βN), heterozygous (β39/βN), and homozygous (β39/β39) nonamplified genomic DNAs. Ratios were obtained by considering Δ% R values after 1200 s of adsorption of conjugated AuNPs. The ratio considers SPRI responses (Δ%R) referred to the PNA-N (Δ%RPNA‑N) and the PNA-M (Δ%RPNA‑M) probes, respectively, when the same DNA target was detected. Homozygous (β39/β39) (mean Δ%RPNA‑N/Δ% RPNA‑M = 0.55, confidence interval at the 99% level CI = 0.55 ( 0.15, replicate measurements n = 10), heterozygous (β39/βN) (mean Δ% RPNA‑N/Δ%RPNA‑M = 0.98, CI = 0.98 ( 0.04, n = 10), and normal (βN/ βN) DNAs (mean Δ%RPNA‑N/Δ%RPNA‑M = 1.4, CI = 1.4 ( 0.2, n = 7) generated significantly different Δ%RPNA‑N/Δ%RPNA‑M ratios (twotailed t test, level 99%, P < 0.0001). Error bars represent the 99% confidence interval (CI) of the mean. The asterisks (/, //) indicate statistically different values. ν represents the degrees of freedom.
the described method can selectively discriminate between normal, β39 homozygous, and β39 heterozygous DNA solutions down to a 2.6 aM concentration (5 pg μL1) by using only about 470 molecules of the genomic DNA. The nanoparticle-enhanced SPRI ultrasensitive detection of genomic DNA here described takes advantage of both the specific properties of PNA probes and the use of properly functionalized AuNPs (see the Experimental Section) and requires an accurate balancing between the number of surface immobilized probes, the number of target molecules, and the conjugated AuNP concentration. Uncharged PNA probes form a neutral layer on the gold surface of the SPRI sensor able to interact with the complementary DNA target more efficiently and with an improved selectivity than traditional oligonucleotide probes.16 The nanoparticle-enhanced SPRI method here described differs from those proposed by other authors because (i) neutral PNAs instead of standard negatively charged oligonucleotides were used as probes;39,40 (ii) biotinylated oligonucleotides immobilized onto streptavidin-conjugated AuNPs instead of thiol-modified oligonucleotides adsorbed onto unmodified AuNPs were used to enhance SPRI signals;40 (iii) large fragments of sheared genomic DNAs instead of synthetic oligonucleotides were used as the target;39,40 (iv) experiments were performed under continuous flow conditions, and kinetic curves were acquired in order to properly check the outcome of each step of the SPRI experiment (i.e., baseline control, PNAs immobilization, genomic DNA adsorption, and conjugated AuNPs adsorption).39,40 The selective capture and nonspecific adsorption of large ssDNA obtained from the fragmentation and the denaturation of genomic DNA modifies the neutral surface environment obtained after PNA immobilization: ssDNAs are negatively charged and highly rich in nucleobase residues and polar groups. The interaction of properly functionalized AuNPs
ARTICLE
with the ssDNA-modified surface strongly enhances the SPRI signal; however, the nanoparticle concentration had to be lower than that used for oligonucleotides14 (0.1 vs 1 nM) in order to avoid saturation of the SPRI detected signal. The stabilization of conjugated AuNP suspensions is obtained when the interparticle repulsive and attractive forces are balanced.41 Negatively charged bound oligonucleotides and partially available citrate groups contribute to the repulsive interactions, whereas the interactions between the negatively charged groups and positively charged sites on streptavidin molecules mainly generate attractive forces between conjugated AuNPs. Alteration of the charge-balancing condition leads to AuNP aggregation.42 As far as conjugated AuNPs are concerned, aggregation is induced by both cross-linking and non-cross-linking mechanisms driven by interparticle bonding formation and changes in surface properties, respectively.43 In our case, conjugated AuNPs that diffuse toward the modified SPRI surface interact with large ssDNA fragments carrying negatively charged groups. The interface environment is expected to alter the force balance of suspended conjugated AuNPs, leading to their surface aggregation (Figure 2e). The presence of a limited number of conjugated AuNPs specifically adsorbed, as a consequence of the hybridization reaction between the biotinylated DNAβ3911-mer oligonucleotide present on AuNPs surface (Figure 1) and the complementary target sequence, is expected to increase the number of initial AuNP aggregates and favors the SPRI signal discrimination between target and nontarget DNA sequences.
’ CONCLUSION This study represents the first demonstration of ultrasensitive detection of point mutation using nonamplified genomic DNA isolated from patients affected by an hereditary genetic disease. The strategy described in this report offers advantages over many existing sensing methodologies for the detection of point mutations. First, our approach allows the direct detection of mutations without the need of PCR or other surface enzymatic amplification reactions: we have shown that homozygous or heterozygous DNA samples carrying the β39 point mutation are discriminated both from each other and from normal DNA samples. Second, our approach is convenient, requiring only sonication and denaturation of the genomic DNA samples. The simple procedures minimize the possibility to contaminate the DNA samples to be analyzed. Third, the detection of DNA is ultrasensitive: we achieved attomolar sensitivity for normal, homozygous β39, and heterozygous β39 DNA detection, comparing very favorably with previously reported methods.4,44 The reduced number of genomic DNA equivalents required for the analysis (about 470) allows us to propose the method we describe as a potential new tool for the analysis of rare cell populations. In particular, as a first example, the proposed strategy can be used for achieving a noninvasive prenatal genetic diagnosis based on the analysis of circulating cell-free fetal DNA in maternal blood, thus contributing to the replacement of most of the other currently used approaches requiring invasive procedures such as amniocentesis, chorionic villous sampling, or fetal blood sampling, each associated to a small but finite risk of fetal loss.45 As a second example, our strategy might be useful to detect the mutated genomic DNA in rare circulating tumor cells, allowing improvements in tumor staging and patient selection for targeted therapy. The task of detecting a few mutated cells in 8716
dx.doi.org/10.1021/ac2021932 |Anal. Chem. 2011, 83, 8711–8717
Analytical Chemistry the presence of a large excess of wild-type cells requires highly sensitive and selective assays. Furthermore, the performance of few PCR cycles combined with our ultrasensitive detection approach may allow, possibly, the detection of genetic mutations in single cells or small cell clusters. Manipulation and characterization of single cells is one of the most relevant issues in biotechnology. Our strategy opens new avenues in this specific field, allowing us to propose the possibility of performing prefertilization diagnosis on the oocyte polar body.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected] (G.S.);
[email protected] (R.G.).
’ ACKNOWLEDGMENT The authors thank J. Dostalek, M. R. Lockett, F. Mavilio, P. M. Milos, M. Minunni, J. Yakhmi, and S. Weibel for critical reading of the manuscript. We acknowledge support from MIUR (PRIN 2009 n 20093N774P, FIRB RBRN07BMCT) and Ministero della Sanita (GR-2009-1596647). R.G. is supported by Telethon (contract GGP10214) and by Fondazione CARIPARO (project 2010-2015). ’ REFERENCES (1) The International HapMap Consortium. Nature 2007, 449, 851861. (2) Hindorff, L. A.; Sethupathy, P.; Junkins, H. A.; Ramos, E. M.; Mehta, J. P.; Collins, F. S.; Manolio, T. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 9362–9267. (3) Fan, H. C.; Wang, J.; Potanina, A.; Quake, S. R. Nat. Biotechnol. 2001, 29, 51–57. (4) Ragoussis, J. Annu. Rev. Genomics Hum. Genet. 2009, 10, 117– 133. (5) Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R., III; Smith, T. F. Clin. Microbiol. Rev. 2006, 19, 165–256. (6) Zhang, C.; Xing, D. Chem. Rev. 2010, 110, 4910–4947. (7) Zhou, X.; Xing, D.; Tang, Y.; Chen, W. R. PLoS One 2009, 4, e8074. DOI: 10.1371/journal.pone.0008074. (8) Ho, H. A.; Dore, K.; Boissinot, M.; Bergeron, M. G.; Tanguay, R. M.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2005, 127, 12673–12676. (9) Storhoff, J. J.; Lucas, A. D.; Garimella, V.; Bao, Y. P.; Muller, U. R. Nat. Biotechnol. 2004, 22, 883–887. (10) Chen, Y.; Shortreed, M. R.; Olivier, M.; Smith, L. M. Anal. Chem. 2005, 77, 2400–2405. (11) Lee, H. J.; Wark, A. W.; Corn, R. M. Langmuir 2006, 22, 5241– 5250. (12) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. (13) Zanoli, L. M.; D’Agata, R.; Spoto, G. Anal. Bioanal. Chem. [Online early access]. DOI: 10.1007/s00216-011-5318-3. Published Online: Aug 25, 2011. http://www.springerlink.com/content/5246r72032327g22/. (14) D’Agata, R.; Corradini, R.; Grasso, G.; Marchelli, R.; Spoto, G. ChemBioChem 2008, 9, 2067–2070. (15) D’Agata, R.; Corradini, R.; Ferretti, C.; Zanoli, L.; Gatti, M.; Marchelli, R.; Spoto, G. Biosens. Bioelectron. 2010, 25, 2095–2100.
ARTICLE
(16) Gambari, R. Curr. Pharm. Des. 2001, 7, 1839–1862. (17) Zanoli, L.; D’Agata, R.; Spoto, G. Minerva Biotecnol. 2008, 20, 165–174. (18) Rothenhausler, B.; Knoll, W. Nature 1988, 332, 615–617. (19) D’Agata, R.; Grasso, G.; Iacono, G.; Spoto, G.; Vecchio, G. Org. Biomol. Chem. 2006, 4, 610–612. (20) Arena, G.; Contino, A.; Longo, E.; Sgarlata, C.; Spoto, G.; Zito, V. Chem. Commun. 2004, 16, 1812–1813. (21) Grasso, G.; D’Agata, R.; Zanoli, L.; Spoto, G. Microchem. J. 2009, 93, 82–86. (22) Kyo, M.; Usui-Aoki, K.; Koga, H. Anal. Chem. 2005, 77, 7115– 7121. (23) Traeger-Synodinos, J.; Vrettou, C.; Kanavakis, E. Expert Rev. Mol. Diagn. 2011, 11, 299–312. (24) Minunni, M.; Tombelli, S.; Scielzi, R.; Mannelli, I.; Mascini, M.; Gaudiano, C. Anal. Chim. Acta 2003, 481, 55–64. (25) Lo, Y. M. D.; Chan, K. C. A.; Sun, H.; Chen, E. Z.; Jiang, P.; Lun, F. M. F.; Zheng, Y. W.; Leung, T. Y.; Lau, T. K.; Cantor, C. R.; Chiu, R. W. K. Sci. Transl. Med. 2010, 2, 61ra91. (26) Corradini, R.; Feriotto, G.; Sforza, S.; Marchelli, R.; Gambari, R. J. Mol. Recognit. 2004, 17, 76. (27) Feriotto, G.; Breveglieri, G.; Finotti, A.; Gardenghi, S.; Gambari, R. Lab. Invest. 2004, 84, 796–803. (28) Feriotto, G.; Ferlini, A.; Ravani, A.; Calzolari, E.; Mischiati, C.; Bianchi, N.; Gambari, R. Hum. Mutat. 2001, 18, 70–81. (29) Feriotto, G.; Breveglieri, G.; Gardenghi, S.; Carandina, G.; Gambari, R. Mol. Diagn. 2004, 8, 33–41. (30) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735–743. (31) Haiss, W.; Thanh, N. T. K.; Aveyard, J.; Fernig, D. G. Anal. Chem. 2007, 79, 4215–4221. (32) Grasso, G.; Fragai, M.; Rizzarelli, E.; Spoto, G.; Yeo, K. J. J. Mass Spectrom. 2006, 41, 1561–1569. (33) Grasso, G.; D’Agata, R.; Rizzarelli, E.; Spoto, G.; D’Andrea, L.; Pedone, C.; Picardi, A.; Romanelli, A.; Fragai, M.; Yeo, K. J. J. Mass Spectrom. 2005, 40, 1565–1571. (34) Jung, L. S.; Campbell, C. T.; Chinowsky, T. M.; Mar, M. N.; Yee, S. S. Langmuir 1998, 14, 5636–5648. (35) Shumaker-Parry, J. S.; Campbell, C. T. Anal. Chem. 2004, 76, 907–917. (36) Park, H.; Germini, A.; Sforza, S.; Corradini, R.; Marchelli, R.; Knoll, W. Biointerphases 2007, 2, 80–88. (37) Mann, T. L.; Krull, U. J. Biosens. Bioelectron. 2004, 20, 945–955. (38) International Human Genome Sequencing Consortium. Nature 2004, 431, 931945. (39) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071–9077. (40) Fang, S. P.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006, 128, 14044–14046. (41) Sujit, K. G.; Tarasankar, P. Chem. Rev. 2007, 107, 4797–4862. (42) Kim, T.; Lee, K.; Gong, M.-S.; Joo, S.-W. Langmuir 2005, 21, 9524–9528. (43) Zhao, W.; Brook, M. A.; Li, Y. F. ChemBioChem 2008, 9, 2363–2371. (44) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253–257. (45) Lo, Y. M. D.; Chiu, R. W. K. Nat. Rev. Genet. 2007, 8, 71–77.
8717
dx.doi.org/10.1021/ac2021932 |Anal. Chem. 2011, 83, 8711–8717
