Point Mutation Detection by Surface Plasmon Resonance Imaging

23 Jan 2008 - The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this ...
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Anal. Chem. 2008, 80, 1049-1057

Point Mutation Detection by Surface Plasmon Resonance Imaging Coupled with a Temperature Scan Method in a Model System J. B. Fiche,† J. Fuchs, A. Buhot,* R. Calemczuk, and T. Livache

SPrAM UMR 5819 (UJF, CNRS, CEA), DRFMC, CEA Grenoble, France

The detection of point mutations in genes presents clear biological and medical interest. Various methods have been considered. In this paper, we take advantage of surface plasmon resonance imaging, a technique allowing detection of unlabeled DNA hybridization. Coupled with a temperature scan, this approach allows us to determine the presence of single-point mutations in oligonucleotide samples from the analysis of DNA’s melting curves in either the homozygous or heterozygous case. Moreover, these experimental data are confirmed in good agreement with numerical calculations. During the last 3 decades, our knowledge concerning DNA mutations and their consequences on human beings has considerably increased. First, we know they can either promote genetic disease like cystic fibrosis1 or, on the contrary, act as a protection against several pathologies like AIDS.2,3 In the same way, it is now commonly admitted that mutations of specific kinds of genesstumor suppressor genes (TSG) and proto-oncogenes particularlyscan increase the risk of cancer. As a nonexhaustive list we can cite BRCA1 for breast cancer,4 P53 gene as an example of TSG5 or K-ras, whose mutations have been used to the purpose of this work.6 At last, with the human genome project, a considerable amount of work has been focused on the detection of single-nucleotide polymorphisms (SNPs) since they represent the most common and widespread form of mutations.7 SNPs can obviously be involved in genetic diseases or cancers, but several other properties are of great interest. Indeed, they should be useful * To whom correspondence should be addressed. Phone: +33 438 78 38 68. Fax: +33 438 78 56 91. E-mail: [email protected]. † Current address: Department of Physics, University of Guelph, Canada. (1) Kerem, B.-S.; Rommens, J. M.; Buchanan, J. A.; Markiewicz, D. T.; Cox, K.; Chakravarti, A.; Buchwald, M.; Tsui, L.-C. Science 1989, 245, 1073-1080. (2) Samson, M.; Libert, F.; Doranz, B. J.; Rucker, J.; Liesnard, C.; Farber, C.M.; Saragosti, S.; Lapoume´roulie, C.; Cognaux, J.; Forceille, C.; Muyldermans, G.; Verhofstede, C.; Burtonboy, G.; Georges, M.; Imai, T.; Rana, S.; Yi, Y.; Smyth, R. J.; Collman, R. G.; Doms, R. W.; Vassart, G.; Parmentier, M. Nature 1996, 382, 722-725. (3) Hladik, F.; Liu, H.; Speelmon, E.; Livingston-Rosanoff, D.; Wilson, S.; Sakchalathorn, P.; Hwangbo, Y.; Greene, B.; Zhu, T.; McElrath, M. J. J. Virol. 2005, 79, 11677-11684. (4) Rosen, E. M.; Fan, S.; Isaacs, C. Endocrine-Relat. Cancer 2005, 12, 533548. (5) Efeyan, A.; Garcia-Cao, I.; Herranz, D.; Velasco-Miguel, S.; Serrano, M. Nature 2006, 443, 159-159. (6) Guerrero, S.; Casanova, I.; Farre´, L.; Mazo, A.; Capella`, G.; Mangues, R. Cancer Res. 2000, 60, 6750-6756. (7) The International SNP Map Working Group. Nature 2001, 409, 928-933. 10.1021/ac7019877 CCC: $40.75 Published on Web 01/23/2008

© 2008 American Chemical Society

to understand the mechanisms involved in the differentiation between genotype and phenotype and could also be used as genetic markers to study and understand the human evolution.8,9 Considering the biological and medical impacts of DNA mutations, many methods have been dedicated to their detection. On one hand, we have methods relying on electrophoresis detection as, for example, denaturant and temperature gradient gel electrophoresis,10 single-strand conformation polymorphisms,11 or heteroduplex analysis.12 These methods have recently been improved using capillary or microchip detection which potentially allows the analysis of hundreds of samples in a few minutes.12-14 On the other hand, other methods can be used in solution, during or right after the PCR amplification step. This form of detection, also known as quantitative PCR, uses various approaches like molecular beacons,15,16 scorpion primers,17,18 TaqMan polymerase19 or LightCycler melting curve analysis.20,21 These techniques are generally greatly appreciated for their simplicity and sensitivity but, as a drawback, still remain quite difficult to parallelize. At last, since the last 20 years, numerous works have demonstrated the potential of DNA chips to detect thousands of point mutations at the same time.9,22 However, the detection using fluorescent dyes is not the only way to detect hybridization on a surface. Several (8) Chakravarti, A. Nature 2001, 409, 822-823. (9) Kennedy, G. C.; Matsuzaki, H.; Dong, S.; Liu, W.-M.; Huang, J.; Liu, G.; Su, X.; Cao, M.; Chen, W.; Zhang, J.; Liu, W.; Yang, G.; Di, X.; Ryder, T.; He, Z.; Surti, U.; Phillips, M. S.; Boyce-Jacino, M. T.; Fodor, S. P.; Jones, K.W. Nat. Biotechnol. 2003, 21, 1233-1237. (10) Sheffield, V. C.; Cox, D. R.; Lerman, L. S.; Myers, R. M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 232-236. (11) Orita, M.; Iwahana, H.; Kanazawa, H.; Hayashi, K.; Sekiya, T. Proc. Natl. Acad. Sci. 1989, 86, 2766-2770. (12) Tian, H.; Brody, L. C.; Landers, J. P. Genome Res. 2000, 10, 1403-1413. (13) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-2186. (14) Landers, J. P. Anal. Chem. 2003, 75, 2919-2927. (15) Tyagi, S.; Marras, S. A. E.; Kramer, F. R. Nat. Biotechnol. 2000, 18, 11911196. (16) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (17) Whitcombe, D.; Theaker, J.; Guy, S. P.; Brown, T.; Little, S. Nat. Biotechnol. 1999, 17, 804-807. (18) Thelwell, N.; Millington, S.; Solinas, A.; Booth, J.; Brown, T. Nucleic Acids Res. 2000, 28, 3752-3761. (19) Lee, L. G.; Connell, C. R.; Bloch, W. Nucleic Acids Res. 1993, 21, 37613766. (20) Millward, H.; Samowitz, W.; Wittwer, C. T.; Bernard, P. S. Clin. Chem. 2002, 48, 1321-1328. (21) Wittwer, C. T.; Reed, G. H.; Gundry, C. N.; Vandersteen, J. G.; Pryor, R. J. Clin. Chem. 2003, 49, 853-860. (22) Gresham, D.; Ruderfer, D. M.; Pratt, S. C.; Schacherer, J.; Dunham, M. J.; Botstein, D.; Kruglyak, L. Science 2006, 311, 1932-1936.

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other methods have been proposed in the past 15 years. Among them, and as a nonexhaustive list, we can point out techniques like surface plasmon resonance,23-26 microcantilevers,27,28 or electrochemistry.29 In the present work, we demonstrate the potential application of surface plasmon resonance (SPR) imaging for the detection of point mutations on DNA chips by melting curve analysis in nonequilibrium conditions. SPR is a label-free technique used to detect a large range of biological interactions occurring on a surface.30-32 Biological processes like DNA/DNA hybridization or DNA/protein interactions can be detected by simply measuring the change of reflectivity as function of time,23-26,33-35 giving access to real-time kinetics. Moreover, the imaging option (SPRi) enables parallel analysis using DNA chip format. This is particularly attractive for mutation screening since the properties of many sequences can be compared during the same target injection. This translates into faster experiments with fewer sample injection needed. Consequently, SPRi is of great interest for point mutation detection as a low-cost technique allowing the detection of hybridization in real time, on a DNA chip format and without the need for labeled targets. In this paper, the principle of detection lies in the comparison between the melting behaviors of perfectly matched and mismatched duplexes. Such detection protocol has been previously used by several groups,36-44 but to the best of our knowledge, all these examples used fluorescence as the (23) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (24) Guedon, P.; Livache, T.; Martin, F.; Lesbre, F.; Roget, A.; Bidan, G.; Le´vy, Y. Anal. Chem. 2000, 72, 6003-6009. (25) Wolf, L. K.; Fullenkamp, D. E.; Georgiadis, R. M. J. Am. Chem. Soc. 2005, 127, 17453-17459. (26) Fiche, J. B.; Buhot, A.; Calemczuk, R.; Livache, T. Biophys. J. 2007, 92, 935-946. (27) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Gu ¨ ntherodt, H.-J.; Gerber, Ch.; Gimzewski, J. K. Science 2000, 288, 316318. (28) McKendry, R.; Zhang, J.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Gu ¨ ntherodt, H.-J.; Gerber, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9783-9788. (29) Komatsu, M.; Yamashita, K.; Uchida, K.; Kondo, H.; Takenaka, S. Electochim. Acta 2006, 51, 2023-2029. (30) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569-638. (31) Smith, E. A.; Corn, R. M. Appl. Spectrosc. 2003, 57, 320A-332A. (32) Rich, R. L.; Myszka, D. G. Anal. Biochem. 2007, 361, 1-6. (33) Maillart, E.; Brengel-Pesce, K.; Capela, D.; Roget, A.; Livache, T.; Canva, M.; Le´vy, Y.; Soussi, T. Oncogene 2004, 23, 5543-5550. (34) Wegner, G. J.; Wark, A. W.; Lee, H. J.; Codner, E.; Saeki, T.; Fang, S.; Corn, R. M. Anal. Chem. 2004, 76, 5677-5684. (35) Lee, H. J.; Wark, A. W.; Goodrich, T. T.; Fang, S.; Corn, R. M. Langmuir 2005, 21, 4050-4057. (36) Drobyshev, A.; Mologina, N.; Shik, V.; Pobedimskaya, D.; Yershov, G.; Mirzabekov, A. Gene 1997, 188, 45-52. (37) Khomyakova, E. B.; Dreval, E. V.; Tran-Dang, M.; Potier, M.-C.; Soussaline, F. P. Cell. Mol. Biol. 2004, 50, 217-224. (38) Wick, L. M.; Rouillard, J. M.; Whittam, T. S.; Gulari, E.; Tiedje, J. M.; Hashsham, S. A. Nucleic Acids Res. 2006, 34, e26. (39) Jobs, M.; Howell, W. M.; Stro¨mqvist, L.; Mayr, T.; Brookes, A. J. Genome Res. 2003, 13, 916-924. (40) Mao, H.; Holden, M. A.; You, M.; Cremer, P. S. Anal. Chem. 2002, 74, 5071-5075. (41) Stimpson, D. I.; Hoijer, J. V.; Hsieh, W. T.; Jou, C.; Gordon, J.; Theriault, T.; Gamble, R.; Baldeschwieler, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 63796383. (42) Watterson, J. H.; Raha, S.; Kotoris, C. C.; Wust, C. C.; Gharabaghi, F.; Jantzi, S. C.; Haynes, N. K.; Gendron, N. H.; Krull, U. J.; Mackenzie, A. E.; Piunno, P. A. E. Nucleic Acids Res. 2004, 32, e18. (43) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (44) Pozhitkov, A. E.; Stedtfeld, R. D.; Hashsham, S. A.; Noble, P. A. Nucleic Acids Res. 2007, 35, e70.

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detection method and only a few of them can be used on DNA chips. Thus, as already suggested by Thiel et al. 10 years ago,23 the use of a label-free technique like SPRi is advantageous by simplifying the preparation of the sample and preventing all the drawback effects linked to fluorescence. In the following, we demonstrate that SPRi can be used to detect point mutations in the model case of oligonucleotide targets hybridized on DNA chips in both homozygous and heterozygous cases, an indispensable condition for SNP detection. Moreover, this technique displays an interesting flexibility since the detection was successfully tested for two sets of mutations without the need of specific optimization. At last, results are in good agreement with theoretical predictions from the nearest-neighbor model (NN),45,46 offering an interesting way to control the validity of the results. EXPERIMENTAL SECTION DNA Sequences. All oligonucleotide (ODN) sequences used in this study are listed in Table 1. The probes were synthesized by ApiBio (Biomerieux, France). They bear on their 5′ end a 10 thymine spacer and a pyrrole (Py) moiety needed for the electropolymerization grafting method. The probes are 15 bases long with an average GC/AT ratio of 75%. Theoretical calculations with the NN model45,46 predicted a melting temperature for perfectly matched duplexes below 70 °C which allows us to study the denaturation on a temperature range from 25 to 70 °C. The probes are called N for the wild type and Mi for the mutated ones, with i ) 1-6. The mutation is always placed in the middle of the sequence to increase the destabilizing effect on the duplexes. We study several mutations of the K-ras gene as previously described in the literature,6,47,48 and we consider two positions for the mutations: on the eighth base (M1, M2, and M3) and on the ninth base (M4, M5, and M6). The other probe, PC, is used as positive control. The DNA targets were synthesized in our laboratory and purified by HPLC. Four different sequences were used: T3, T4, and T5 are 15-mer sequences hybridizing perfectly with M3, M4, and M5, respectively. T is perfectly matched with the positive control PC. All sequences are presented in Table 1, and a color code will be used in the following to simplify the interpretation of the results. To demonstrate that the efficiency of our method is not dependent on the nature of the Mi sequences, we repeat the experiment using another set of sequences. In that case, the mutation is a SNP located on the gene coding for the cycline D1 protein (SNP A870G from the 242th codon, exon 4). The choice of this protein is also motivated by its biologically relevant role in carcinogenesis since it is involved in cell-cycle regulation and DNA transcription mechanisms. Both probes and targets are listed in Table 1 and were synthesized and purified in our laboratory. The mutation is now located on the ninth base from the 5′ end of the 16 base probe and consists in the substitution of an adenine base (ASOCCND1s-A) by a guanine (ASO-CCND1s-G). Two different (45) SantaLucia, J., Jr. Proc. Natl. Acad. Sci: U.S.A. 1998, 95, 1460-1465. (46) SantaLucia, J., Jr.; Hicks, D. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415-440. (47) Lopez-Crapez, E.; Livache, T.; Marchand, J.; Grenier, J. Clin. Chem. 2001, 47, 186-194. (48) Cerottini, J.-P.; Caplin, S.; Saraga, E.; Givel, J.-C.; Benhattar, J. Am. J. Surg. 1998, 175, 198-202.

Table 1. Oligonucleotide Sequences Used for the Point Mutation Detectiona

a

A color is associated to each probe to simplify the interpretation of the melting curves.

targets are used, TA and TG, which perfectly match ASOCCND1s-A and ASO-CCND1s-G probes, respectively. Preparation of the DNA Chips. The DNA chips were prepared on a glass prism (high refractive index n ) 1.7) coated with a gold layer (thickness 50 nm). First, the gold layer was immersed in a solution containing ethanol and dodecanethiol for 10 min at room temperature in order to create a hydrophobic thiol monolayer. Then, the prism was rinsed with ethanol and deionized water (18.2 MΩ‚cm). Finally, the DNA spots were fabricated using the pyrrole copolymerization as previously described.49 Briefly, for each probe, we prepared an electropolymerization solution containing 20 mM of pyrrole and 10 µM of pyrrole-modified ODN (Table 1). The probes were then addressed on the thiolated surface with a micropipet, and the grafting is performed by applying an electric pulse of 2 V for 100 ms. The grafting process is carried out using a difference of potential of 2 V between the counter electrode (tip) and the gold surface in presence of pyrrole monomers. The oxidization of this monomer occurs at a potential of ∼0.7 V versus a saturated calomel electrode. At this potential, the oxidization of DNA bases cannot occur. The polypyrrole spots obtained with this method were around 400 µm of diameter and have a thickness below 5 nm. From this protocol, the density of probes was estimated to be around 10 pmol‚cm-2.24 By successive copolymerization steps, the probes were arrayed in different spots on the prism. Polypyrrole (Ppy) spots without ODN were used as a negative control, whereas spots grafted with the probe PC served as a positive control. Each probe was grafted three times (three different spots) in order to control the reproducibility and the dispersion due to the electropolymerization process on the reflectivity signal and the melting curves. The DNA chips were dried and stored at 4 °C under argon between each use and can be used for more than 25 temperature scan experiments without significant decrease on the hybridization signal. SPR Imaging Setup and Temperature Control Device. An SPRi apparatus from GenOptics (Orsay, France) with an incoher(49) Livache, T.; Guedon, P.; Brakha, C.; Roget, A.; Le´vy, Y.; Bidan, G. Synth. Met. 2001, 121, 1443-1444.

ent light source (λ ) 635 nm) is used to follow the hybridization kinetics as described previously.26 This optical method is sensitive to small changes of the refractive index near the gold layer (around 200 nm in depth) (Figure 1). The SPR images are captured by a CCD camera and a LabView software (GenOptics, France) allows for real-time averaging of the intensity on each spot in order to obtain the reflectivity signals. All hybridization reactions were done in a homemade heated flow cell of 4 µL (Figure 1). The cell temperature was measured using a negative temperature coefficient resistor (NTC) and regulated by electrical heating using a PID controller. We are able to regulate the temperature from 25 to 70 °C with 0.05 °C precision. A careful in-line degassing of the solution is needed to avoid bubble formation (Altech Elite). Hybridization and Point Mutation Detection Experiments. The hybridization experiments were done in PBS buffer (Sigma) at 0.35 M NaCl (SDS) prepared with deionized water (18.2 MΩ‚ cm). A continuous flow streams in the cell with a flow rate of 80 µL/min. The hybridization was performed by injecting during 10 min 0.5 mL of target solution with concentrations between 50 and 250 nM. Nonspecific adsorption is not observed on both negative (Ppy spots) and positive controls (spots prepared with the probe PC). The point mutation detection relied on the observation of the melting curves for each probe spotted on the chip. To perform this, a homemade LabView interface controlled the linear increase of temperature in the cell from 25 to 70 °C. The temperature rate was usually fixed at 2 °C/min but could be increased up to 12 °C/min. Since the refractive index of water is strongly dependent on temperature, a slight variation of temperature produced a change of reflectivity around 0.35% per degree. In order to evaluate this contribution we used two temperature scans: the first one was performed before the hybridization reaction and allowed us to measure only the influence of temperature on reflectivity for each spot of the chip. Then, after hybridization had occurred, we performed the second scan. The melting curves were then obtained by subtracting both scans. Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

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Figure 1. Experimental setup: an LED is used as the light source (λ ) 635 nm). The light is polarized in p or s mode by a polarizer and illuminates the functionalized gold layer in contact with the heating flow cell. Reflectivity changes of each spot are followed using a CCD camera monitored by a LabView interface. A degassing system is used to prevent bubble formation in the flow cell during experiments.

It is important to keep in mind that the SPR signal we measure during the temperature scan after hybridization has two different contributions: one due to the change of temperature itself, the other one related to DNA denaturation. The key point is that the signal of the former contribution is generally 10-20 times higher than the one of the latter. Consequently, the scan subtraction described earlier is intended to remove the temperature dependence. It makes sense only if the variation of reflectivity with temperature is highly reproducible. In our case, control spots (Ppy and PC, see Figure 1) are used to assess this point since the scans measured before and after hybridization must be the same, leading to a signal equal to zero after subtraction (data not shown). From the experimental point of view, the reproducibility is obtained by the high stability of our DNA chips, prepared using the Ppy electropolymerization, and several scans performed at the beginning of each experiment to remove the possible contamination of the surface by proteins or other biomolecules. All experiments were performed in nonequilibrium conditions in order to increase the destabilizing effect of mutations on the duplex. Nonequilibrium conditions offer the convenience of not requiring that a constant DNA concentration be provided. In clinical assays, targets are available in small quantities so that the flow conditions would need modification during the detection step. Furthermore, the fact of rinsing with buffer allows to reduce SPRi drawbacks like bubble formation during heating and long-term reflectivity drift. The buffer could then also be changed to apply more stringent conditions using denaturing agents in order to increase point mutation distinction. In order to fulfill nonequilibrium conditions, unbound targets were removed from the cell after each hybridization reaction to ensure a DNA concentration in solution equal to zero during the denaturation experiments. After the 10 min of hybridization at fixed temperature, we wait 5 min with a flow streaming in the cell in order to remove unbound and weakly bound targets from the surface. At the starting temperature of the scan (25 °C), the duplex dissociation is generally sufficiently slow for this 5 min time delay to be negligible. A recent work by Pozhitkov et al.44 showed that mismatched duplexes do not always dissociate before perfect matches. For such sequences the 1052

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detection of the point mutation may impose the determination of the melting temperatures in equilibrium conditions (in the presence of the targets in solution during the temperature scan) and/or of the kinetic parameters for each duplexes. The DNA chip regeneration was achieved by injecting NaOH at 100 mM for 1 min, followed by an injection of deionized water for 3 min. Calculation of Theoretical Melting Curves Using the Nearest-Neighbor Model. Among the numerous models describing DNA hybridization mechanisms, the semiempirical NN model is of great interest. Indeed, it allows an estimation of the thermodynamic parameters ∆H and ∆S of short oligonucleotides (typically less than 20 bases) as a function of their sequence.45,46 Furthermore, those estimations can be performed for either perfectly matched or mismatched duplexes. Considering a concentration of 0.35 M NaCl, ∆H and ∆S were estimated for each duplex obtained from the sets of the probes (N and Mi) and the targets (Ti) (see Table 1) using the free software Hyther (http://ozone3.chem.wayne.edu/cgi-bin/login/ login/showLoginPage.cgi). These values are used to calculate the equilibrium constant K(T) of each duplex as a function of temperature according to the following equation:

(

K(T) ) exp -

∆H - T∆S RT

)

(1)

where T is the temperature in Kelvin and R is the gas constant. As described above, the melting curves are measured in nonequilibrium conditions as unbound targets are removed continuously from the cell after the hybridization process. The time evolution of the fraction θ of hybridized probes on the surface can be calculated assuming the following equation:

dθ(t, T) ) -koff(T)θ(t, T) dt

(2)

where koff is the kinetic rate of denaturation whose value strongly depends on both temperature and duplex sequence. Its value can be estimated as follows:

koff(T) )

kon K(T)

(3)

with kon the kinetic rate of hybridization. In comparison to koff, this rate is almost independent of both temperature and sequence.26,50,51 In other words, the substitution of one base by another in our sequences has only a slight effect on the hybridization rate value. Consequently, we assign the same kon to each probe, and the value of 105 M-1‚s-1 was chosen according to previous results26 and data found in the literature.52,53 The melting curves are numerically determined by solving eq 2 (Mathematica 5.1) using the variation of koff as a function of time-dependent experimental temperature. We assume a linear increase of the temperature with a rate of 2 °C/min. The initial value of θ is fixed to 1 at 25 °C assuming full hybridization of the probes by the targets at such low temperature. When a mixture of different targets is considered, we assume that the same composition is present and hybridized on the spots. This assumption follows from two different arguments: first, the hybridization rates kon are similar when only few mutations are involved preserving the sample’s composition on the spot as in solution, and second, the hybridization time is sufficiently short in order to neglect very slow rearrangements of the targets on the spot favoring the more stable duplex without mutations.50 For a longer waiting time between the hybridization of the targets and the start of the temperature scan, we would expect the ratio between the number of mismatched and perfect match target sequences hybridized to the chip to be closer to the equilibrium one. The various approximations introduced in this calculation (solution-phase thermodynamic parameters, initial conditions, ...) do not allow us to expect a quantitative comparison; however, a qualitative agreement is expected. In particular, the structure of the melting curves and their relative order should be correctly predicted by the theoretical analysis compared to the experimental curves. RESULTS AND DISCUSSION Homozygous Case. The homozygous case is the simplest test for a new point mutation detection method as there are only two possibilities for each probe: either the target in solution brings a mutation or it does not. Most of the detection methods rely on the comparison between mismatched and perfectly matched hybridization amounts at fixed temperature. Indeed, in such techniques, experimental conditions must be optimized in order to selectively increase destabilization effects of mutation on mismatched duplexes. Particular attention must be paid to hybridization and rinsing conditions (temperature, buffer stringency, ...) as well as probe design (length, position of the mutation, GC/AT content, ...). However, such optimizations are strongly dependent on the nature of the sequences grafted on the DNA chip. Hence, for each new mutation, another optimization work must be achieved to find the best condition that allows for the detection of the mutation. Since, in this work, complete melting curves are analyzed, no previous optimization will be necessary. (50) Bishop, J.; Blair, S.; Chagovetz, A. M. Biophys. J. 2006, 90, 831-840. (51) Halperin, A.; Buhot, A.; Zhulina, E. B. Biophys. J. 2005, 89, 796-811. (52) Tawa, K.; Knoll, W. Nucleic Acids Res. 2004, 32, 2372-2377. (53) Okahata, Y.; Kawase, M.; Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288-1296.

Figure 2. Hybridization kinetic curves measured at 30 °C after injection of target T3 at a concentration of 250 nM. The hybridization kinetics for different spots with the same probe sequence (curves with the same color) are presented to illustrate the variability of the response on different spots. Although the spots containing the same probes were prepared in the same conditions, hybridization kinetic curves are not exactly the same (see, for example, M1 in red and M6 in blue). This variability is similar to the difference due to single or double mismatches between probe and target (curves of different colors). The behavior of the perfectly matched probe M3 (yellow curves) and those containing one (purple, red, and orange curves) or two mismatches (green, turquoise, and blue curves) cannot be distinguished. Gray and black curves correspond to positive and negative control spots.

As an example, Figure 2 gives the hybridization kinetics of the target T3 on the DNA chip. In our conditions, we cannot tell which probes contain mutation(s) and which ones do not by simply comparing the hybridization curves. In fact, the destabilizing effect of the mutations is too weak at 30 °C to clearly resolve the sequence of the target. Nevertheless, this is not always true as the SPR signal strongly depends on the target injected. For example, for an injection of the target T5, the effect of one mutation remains hardly detectable, whereas two mutations give rise to a strong decrease of the reflectivity (data not shown). Furthermore, the variation of reflectivity due to the target hybridization not only depends on the probe sequences but also on the spot reproducibility. Indeed, spots prepared with the same probe and grafting conditions do not always display the same equilibrium hybridization signal (see, for example, M4, M6, and N). Consequently, such dispersion makes the discrimination between perfectly matched and mismatched hybridization kinetics difficult. The sequence dependence impairs the detection of mutations using hybridization kinetic profiles. This is even more stringent when different mutations are to be scanned on the same chip. This limitation can be overcome by scanning mutation effects on duplex stability as a function of temperature54 and by comparing the melting behavior of each probe with the target. An example of melting curves obtained by SPRi after the hybridization of the target T3 is shown in Figure 3A. As explained previously (see the Experimental Section), melting curves are obtained in non(54) Halperin, A.; Buhot, A.; Zhulina, E. B. Clin. Chem. 2004, 50, 2254-2262.

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Figure 3. (A) Melting curves measured by SPRi after injection of target T3 at 25 °C and a concentration of 250 nM. The curves can be separated into three families, depending on their behavior when the temperature increases. At low temperature, we first observe the denaturation of all probes containing two mutations (M4, M5, and M6). The denaturation of the probes containing one mutation follows at higher temperature (N, M1, and M2), and we finally observe the denaturation of the perfectly matched duplex, M3-T3. (B) Theoretical predictions for the melting curves using the nearest-neighbor (NN) model. The arrangement of the curves is the same for both the experimental data and the theoretical predictions.

equilibrium conditions as target concentration is null while the temperature scan is performed. Consequently we will no longer refer to the melting temperature Tm since this implies that thermodynamic equilibrium is reached. In the following, we will preferentially use the denaturation temperature Td as an equivalent of Tm for nonequilibrium experiments. As expected, we observe in Figure 3A three different behaviors depending on the amount of mutated bases on each spot. Denaturation of duplexes carrying two mutations is first observed around Td ) 37 °C, which corresponds to the probes M4, M5, and M6 hybridized with the target T3. Then, destabilization of duplexes with one mutation (N, M1, and M2) occurs at higher temperatures. We finally observe the melting of the perfectly matched duplex, M3-T3, at Td ) 53 °C. Clearly, SPRi coupled with temperature scan is a useful technique for point mutation 1054

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detection on DNA chips. The simple recording of the melting curves for each probe allows us to determine the target sequence injected. To the best of our knowledge, this is the first time that such a method is performed with SPRi, and more generally with a label-free method of detection on a DNA chip. This method has a valuable advantage on other techniques relying on the comparison between mismatched and perfectly matched hybridization amounts at a fixed temperature since different mutations may be scanned on the same chip without further optimization steps. The detection by temperature scans remains possible as long as a sufficient amount of hybridization is detected during target injection. In our case, the lowest hybridization signal required for detection is 0.2% (data not shown). This corresponds to a signalto-noise ratio of around 10 and a detection limit for the oligonucleotide target concentrations between 20 and 100 nM, depending on the quality of the DNA chip. As highlighted by Figure 2, the variation of reflectivity due to target hybridization not only depends on the probe sequences but on the spot reproducibility, too. However, this dispersion has no consequence on the temperature detection when systematic renormalization of the melting curves is performed. We normalized the reflectivity for each spot in such a way that the highest and lowest values equal one and zero, respectively. Consequently, melting curves measured for the same probe but on three different spots of the DNA chip overlay almost perfectly after renormalization (see Figure 3A). We can thus conclude that the grafting irreproducibility only affects the sensitivity of the spotssand therefore the variation of reflectivity during hybridizationsbut not the stability of the duplex with temperature. As a consequence, we measured for each probe the denaturation temperature Td with a precision higher than (0.5 °C. This is sufficient to detect a variation of Td as a function of the nature of the mutation as illustrated in Figure 3A for the probes M1, M2, and N. The C-A mismatch (M2) seems to be the most destabilizing with a denaturation temperature of 42.4 ( 0.5 °C and, on the contrary, the G-A mismatch (N) is the most stable with Td ) 48.7 ( 0.5 °C. These results are well reproduced not only for different target injections and different spots on the same chip but also for different chips. In fact, such behavior is an interesting illustration of the consequence of stacking interactions on the duplex stability as there is no hydrogen bond between mismatched bases. Thus, our results could be partially understood taking into account the stacking interaction dependence on the surface of overlap between neighboring bases.55 For example, cytosine (C) and thymine (T) are part of the pyrimidine base family and display only one aromatic ring instead of guanine (G) which displays, as a purine, two aromatic rings. In these conditions, stacking interactions are stronger when replacing the thymine by a guanine than by a cytosine. Consequently, duplexes containing a G-A mismatch are more stable than those containing a C-A mismatch. The normalization procedure of the melting curves reflecting the fraction of hybridized probes on the spot at a given temperature allows us to compare our experimental results to the theoretical predictions. To this purpose, we estimate the thermodynamic parameters ∆H and ∆S of each probe using the NN model45,46 and calculate the corresponding melting curves (see (55) Guckian, K. M.; Schweitzer, B. A.; Ren, R. X.-F.; Sheils, C. J.; Tahmassebi, D. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 2213-2222.

the Experimental Section). Our experimental observations and the theoretical predictions are in good qualitative agreement as the same arrangement of the curves is observed on both parts A and B of Figure 3. The same trends are also observed for the melting curves registered after injections of targets T4 and T5 (data not shown). Only the probe M4 displays in several cases an unexpected behavior, but no convincing argument has been found to explain this inadequacy between theory and experiments. The point to be stressed here, however, is that our results agree with the theoretical predictions which in turn confirm the composition of the sample injected on the chip. Note that the observation of systematically broader melting curves on DNA chips than in theory has been discussed in detail in a previous work.26 It is interpreted as the effect of probes not fully accessible on the spots. The shift in denaturation temperature Td may also be due to the fact that the thermodynamic parameters considered for the theoretical curves correspond to the hybridization reaction in solution and to the fact that the DNA chip format has not been taken into account. Different models have shown possible modifications of the hybridization properties due to the grafting of the probes on a surface (electrostatic interactions, effects of the probe, the target and the spacer chain lengths, ...).51,56,57 In conclusion, SPRi detection coupled with temperature scan and melting curve analysis provides a powerful tool for point mutation detection in the homozygous case. Indeed, regardless of the target considered, differences between the melting processes of both perfectly matched and mismatched duplexes are clearly detected by their difference in denaturation temperatures. For the sequences considered, the sensitivity is sufficient to observe how the nature of the mutation affects the stability of the duplex through stacking interactions. In this case, our results are found in good agreement with theoretical predictions in solution using the NN model. In addition, as illustrated in Figure 4, this method was successfully used for another set of sequence without optimization of the experimental conditions. As suggested by a recent work,43 this may not be true for all sequences. The point mutation detection remains possible, even for temperature rates of 12 °C/min which substantially reduce the time of a temperature scan. Note that there is a slight translation (∼3-4 °C) of the melting curves toward higher temperatures by increasing the scan rate. In fact, this trend is an expected consequence of our nonequilibrium conditions. Indeed the denaturation process is only controlled by the denaturation rate constant koff(T) as the target concentration is null in the cell (see the Experimental Section). Heterozygous Case. The fact that point mutation detection can be performed by SPRi in the homozygous case is not sufficient to think of an eventual medical application. Indeed, even for SNPs, the detection must be efficient in both the homozygous and heterozygous case. As with the DASH method used on DNA chips,39 we take advantage of the melting curve observation to perform the point mutation detection in the heterozygous case. As discussed previously, both sequences present in the solution in equimolar quantity hybridize indifferently with each probe at low temperature. Consequently, each spot of the DNA chip carries two different duplexes and their proportion is determined by the (56) Halperin, A.; Buhot, A.; Zhulina, E. B. Biophys. J. 2004, 86, 718-730. (57) Halperin, A.; Buhot, A.; Zhulina, E. B. J. Phys.: Condens. Matter 2006, 18, S463-S490.

Figure 4. Melting curves obtained with the cycline-D1 sequences using the same experimental conditions as for Figure 3A. Blue curves correspond to the probe ASO-CCND1s-A and green ones to ASOCCND1s-G (see Table 1). The figure displays two different sets of melting curves: the first one with a temperature scan rate of 4 °C/ min (full lines) and the second one with a temperature scan rate of 12 °C/min (dotted lines). The shift due to the change of the speed is linked to our nonequilibrium condition.

composition of the target mixture if the association rates kon are similar. As discussed in a recent paper,50 such behavior impairs the detection of mutations as the thermodynamic equilibrium is reached only after many hours. Indeed, the hybridization behavior can be described in two phases: first, both targets hybridize independently on the spot, and then, duplexes carrying a mutation are slowly replaced by perfectly matched ones, thermodynamically more stable. This latter phase is characterized by a very slow time scale and is the limiting step of the hybridization process with target mixtures. In our case, the fact that both targets hybridize on the same spot is not a problem and may even be used to our advantage for the detection of heterozygous mutations. As illustrated in Figure 5A for a mixture of targets T4 and T5, we observe a two-step denaturation on the spots containing the probe M5 (light-blue curve). This behavior is easily understood by considering that there are two different duplexes on this spot: M5/T5 without mutation and M5/T4 carrying one mutation. Hence, by increasing the temperature, we first observe the denaturation of the mismatched duplexes at low temperature (from 35 to 45 °C) followed by the denaturation of the perfectly matched one for temperatures higher than 55 °C. Although this two-step melting process characterizes the presence of two different targets in the sample, a condition must be fulfilled to observe it. The difference of denaturation temperature ∆Td between both duplexes must be sufficient to allow the observation of a plateau. For example, in Figure 5A, a clear plateau can be observed for the probe M5 as ∆Td ∼ 11 °C between M5/T5 and M5/T4. On the contrary, we only measure an average melting curve for the probe M4 (green) since ∆Td between M4/T4 and M4/T5 is lower than 5 °C. Similarly to the homozygous case, the melting behavior is predicted using the NN model for a mixture of targets. As we can see in Figure 5B, theoretical curves displayed the same arrangement as the experimental data and a plateau is only Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

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Figure 6. Derivative curves calculated using the experimental data collected from the heterozygous case T4 + T5 (see Figure 5). For each probe, the derivative curves are calculated using the average of the melting curves measured on three spots with identical probes of one DNA chip. The plateau observed for the probe M5 appears in a well-defined two-peak structure, clearly indicating the presence of the different duplexes on the surface and, thus, the two different targets in the analyzed sample.

Figure 5. (A) Melting curves obtained after hybridization of an equimolar mixture of the targets T4 and T5 at 125 nM. A plateau is observed for the probe M5, illustrating the two-step melting behavior in the heterozygous case: first, at low temperature, the denaturation of the mismatched duplex M5-T4 and then, at higher temperature, the denaturation of the perfectly matched duplex M5-T5. No plateau is observed for the other probes as the difference of Td between perfectly matched and mismatched duplexes is too small. (B) Theoretical predictions for the melting curves using the nearestneighbor (NN) model. The arrangement of the curves is the same for both the experimental data and the theoretical predictions.

predicted for the probe M5. Thus, as previously discussed, theoretical predictions based on the NN model constitute a useful way to control our experimental results and to confirm the content of the analyzed sample. With the point mutation detection in mixtures of targets like the heterozygous case, the denaturation temperature Td cannot be used in an easy way to determine the composition of the sample due to the presence of the plateau. Derivative curves are a more useful way as two-step melting curves lead to the observation of well-defined two-peak structure, as illustrated in Figure 6. The distinction between both homozygous and heterozygous cases depends on the number of observed peaks, and the composition of the sample is simply determined by the probes presenting the highest denaturation temperatures (peaks at highest temperature). 1056 Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

Detection Limit. The detection of mixtures of wild-type and mutated DNA becomes even far more complex for somatic point mutations as their proportion in the DNA sample may be arbitrarily low especially when the DNA is extracted from body fluids. The amount of mutated targets must be sufficient to allow the detection. In order to estimate our detection threshold, several experiments were carried out varying the proportion of each target in solution. Our estimation lay in a range between 10% and 25%, depending on the nature of the sequences analyzed. Obviously, these values are higher than those obtained with labeled techniques like electrophoresis12 or minisequencing.58 However, it compares favorably with others label-free techniques like pyrosequencing.59 The detection of somatic point mutations remains a challenge if the proportion of sequences carrying a mutation is lower than 10% in the DNA sample. However, temperature cycles may improve the detection limit by modifying the composition of the targets on the spots compared to the one in solution. Nevertheless, our sensitivity is perfectly sufficient for SNP detection for either homozygous or heterozygous and for somatic point mutations when tumor extracts are considered. CONCLUSION We have presented here a new label-free method for point mutation detection using SPR imaging coupled with temperature scans for melting curve determination. As previously described, such a tool can be used to study hybridization properties on DNA chips and permits an estimation of the kinetic and thermodynamic parameters of each probe immobilized on the surface.26 In the same way, temperature scans can be used to detect point mutations in nonequilibrium conditions by measuring the melting (58) Gerry, N. P.; Witowski, N. E.; Day, J.; Hammer, R. P.; Barany, G.; Barany, F. J. Mol. Biol. 1999, 292, 251-262. (59) Garcia, C. A.; Ahmadian, A.; Gharizadeh, B.; Lundeberg, J.; Ronaghi, M.; Nyre´n, P. Gene 2000, 253, 249-257.

curves of the various duplexes formed on the chip after injection and hybridization of a DNA sample. Our results suggest that this technique is suitable for SNP detection as the composition of the sample can be accurately predicted in both the homozygous and heterozygous case. Moreover, this technique can be used for any type of sequence without specific optimization of the hybridization parameters, conferring to this technique an interesting flexibility. Recent results by Pozhitkov et al.44 show that the mismatch duplexes do not always dissociate faster than the perfect match. For such sequences our method may lead to misinterpretation of the results, and complementary data like equilibrium analysis and kinetic parameters determination might be useful. But, as previously discussed,26 SPRi remains an interesting tool to perform those experiments. We are also able to accurately compare our results to simple theoretical predictions based one the NN model in order to confirm the composition of the sample. The next step of this work consists in the detection of point mutation in biological samples obtained after PCR amplification. The PCR sequences are longer than the synthetic ODN. This

should increase the SPR signal. However, it also promotes the formation of secondary structures which could imply hybridization competition effects. All these differences tend to reduce the speed and the efficiency of the hybridization process. Fortunately, the method based on temperature scans allows the detection in nonequilibrium conditions and is efficient even with a low amount of hybridization. Thus, we think SPR imaging coupled with temperature scan can be an efficient and low-cost tool for point mutation detection on DNA chips. It is important to notice that it may also be of great interest to study other biological systems where temperature plays an important role. ACKNOWLEDGMENT We thank Nanobio for funding support.

Received for review September 21, 2007. Accepted December 4, 2007. AC7019877

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