Langmuir 1997, 13, 701-707
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Adsorption of Single-Stranded DNA Fragments onto Cationic Aminated Latex Particles Franc¸ ois Ganachaud,† Abdelhamid Elaı¨ssari,*,† Christian Pichot,† Ali Laayoun,‡ and Philippe Cros‡ Ecole Normale Superieure de Lyon, 46 Alle´ e d’Italie, 69364 Lyon Cedex 07, France Received September 17, 1996. In Final Form: November 27, 1996X In this study, the adsorption of single-stranded oligodeoxyribonucleotides (ODN) onto well-characterized polymer latex particles has been investigated. At first, the case of poly(thymidylic acid) (dT35) was thoroughly examined as a function of pH and ionic strength in the presence of cationic polystyrene colloids prepared by emulsifier-free copolymerization of styrene and vinylbenzylamine hydrochloride. Due to the polyelectrolyte character of the oligodeoxyribonucleotides (which are negatively charged) and the positive charges of the particles, strong adsorption was clearly noticed together with a high affinity; in addition, a decrease in the maximal adsorption was observed upon raising the pH. These adsorption phenomena corroborate that electrostatic forces play a major role in the adsorption process; however the contribution of hydrophobic forces was also evidenced using the various adsorption isotherms at various pHs and extrapolating these results to zero surface charge density (σ ) 0) and to zero zeta-potential (ζ ) 0) of the latex particles. Moreover, the effect of poly(thymidylic acid) chain length on the maximum adsorbed amount on the latex particles was also investigated, providing information on the state of conformation of the ODN at the particle surface; it was suggested that the ODN adsorbs in a flat conformation. Such a behavior can be extended regardless of ODN nature so long as the support was oppositely charged. Finally, an attempt was made to predict the adsorption behavior of single-stranded ODN using the general approach of Hesselink for polyelectrolytes.
I. Introduction In the field of clinical diagnosis, synthetic oligodeoxyribonucleotides (ODN’s) have become a powerful tool in the detection of pathogenic microorganisms such as viruses, bacteria, and fungi.1-3 For this purpose, oligodeoxyribonucleotides that are bound to solid support are useful as hybridization probes and affinity matrices for binding specifically to complementary target DNA or RNA sequences.4-8 Among some of the major limitations of the hybridization methods on the solid supports, in terms of sensitivity and reproducibility, is that the amount of the oligodeoxyribonucleotide probe which is immobilized on the support is not completely available for hybridization with the target sequence. The hybridization rate of this form of immobilized DNA is slower than that of the corresponding reaction in solution; however, some microspheric supports such as polystyrene latex9-11 particles are found to exhibit excellent hybridization. * To whom correspondence should be addressed at CNRSBioMe´rieux, UMR-103, ENSsLyon, 46 Alle´e d’Italie, 69364 Lyon Cedex 07, France. Tel: (33) 04-72-72-83-64. Fax: (33) 04-72-7285-33. E-mail:
[email protected]. † Unite ´ mixte CNRS-BioMe´rieux. ‡ Laboratoire des sondes nucle ´ iques BioMe´rieux. X Abstract published in Advance ACS Abstracts, February 1, 1997. (1) Hunt, J. M.; Persing, D. H. In DNA Probes: Background, Application, Procedures; Keller, G. H., Manak, M. M., Eds.; Stockton Press: New York, 1993; pp 525-555. (2) Keller, G. H. In DNA Probes: Background, Application, Procedures; Keller, G. H., Manak, M. M., Eds.; Stockton Press: New York, 1993; pp 377-403. (3) English, U.; Gauss, D. H. Angew. Chem. 1991, 30, 613. (4) Gold, L. J. Biol. Chem. 1995, 270, 13581. (5) Symons, R. H., Ed. In Nucleic Acid Probes; CRC Press: Boca Raton, FL, 1989. (6) Gold, L.; Polisky, B.; Uhlenbeck, O.; Yarus, M. Annu. Rev. Biochem. 1995, 64, 763. (7) Walker, G. T.; Linn, C. P.; Nadeau, J. Nucleic Acids Res. 1996, 24, 384. (8) Lamture, J. B.; Beattie, K. L.; Burke, B. E.; Eggers, M. D.; Ehrlich, D. J.; Fowler, R.; Hollis, M. A.; Kosicki, B. B.; Reich, R. K.; Smith, S. R.; Varma, R. S.; Hogan, M. E. Nucleic Acids Res. 1994, 22, 2121. (9) Van Ness, J.; Kalbfleisch, S.; Petrie, C. R.; Reed, M. W.; Tabone, J. C.; Vermeulen, N. M. J. Nucleic Acids Res. 1991, 19, 3345. (10) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679.
S0743-7463(96)00896-7 CCC: $14.00
Previous studies have been performed on the adsorption behavior of nucleic probes onto charged polystyrene particles. At first, a preliminary study was devoted to the adsorption of oligodeoxyribonucleotides containing three nucleotides (T, C, G) and with the following structure (5′-TCG TCG TCG CTG TCT CCG CTT CTT CCT GCC-3′) (dC12G5T10) onto anionically and cationically charged latex particles12 at low ionic strength. Due to the negative charges of the phosphodiesters of the nucleotides, it was found that the adsorbed amount was much larger onto the cationic latexes because of electrostatic attractions. However, the adsorption of ODN fragments onto the anionic latex was not negligible even at low ionic strength (10-3), a result which was interpreted by the influence of nonionic interactions (possibly hydrophobic ones between polystyrene and base units). A second paper reported on the adsorption of fluoresceinlabeled poly(adenylic acid) (FITC-dA30), i.e., homopoly(nucleic acid) containing only adenine nucleotides, onto cationically charged latex particles.13 The main conclusion was that dA30 adsorption was strongly pH-dependent, a maximal value being obtained in the low-pH region. In addition, the contribution of hydrophobic forces was also evidenced, leading to a residual adsorbed amount of the same order of magnitude as with the dC12G5T10 (i.e., 0.300.40 mg m-2). Interestingly, a series of papers by Walker and Grant14-16 also reported on the behavior of various singlestranded DNA molecules (exclusively based on poly(thymidylic acid)) in the presence of anionically or cationically charged latexes. The authors first showed the validity of a biochemical technique called hydroxyl radical footprinting (HRF) to explore the conformation of (11) Day, P. J. R.; Flora, P. S.; Fox, J. E.; Walker, M. R. Biochem. J. 1991, 278, 735. (12) Elaı¨ssari, A.; Cros, P.; Pichot, C.; Laurent, V.; Mandrand, B. Colloids Surf. 1994, 83, 25. (13) Elaı¨ssari, A.; Pichot, C.; Delair, T.; Cros, P.; Ku¨rfurst, R. Langmuir 1995, 11, 1261. (14) Walker, H. W.; Grant, S. B. Langmuir 1995, 11, 3772. (15) Walker, H. W.; Grant, S. B. J. Colloid Interface Sci. 1996, 179, 552. (16) Walker, H. W.; Grant, S. B. Langmuir 1996, 12, 3151.
© 1997 American Chemical Society
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Chart 1. Structure of dT3 Poly(thymidylic acid) Nucleotide Bearing an Aliphatic Primary Amine
Ganachaud et al. Table 1. Latex Recipe for Batch Processa latex code
VBAH (mmol L-1)
V-50 (mmol L-1)
conversion (%)
GAF2 GAF3 GAF1
0.00 2.94 5.88
5.9 5.9 5.9
83 92 96
a
Water, 200 g; styrene, 20 g; temperature, 70 °C; 300 rpm. Table 2. Latex Recipe for Shot-Growth Processa
latex code
VBAH (mmol L-1)
conversion (%)
GAF10 GAF11
14.70 14.70
72 95
a The functionalization took place after 80% conversion of the batch process: styrene, 1.06 mol L-1; V-50 (initiator), 7.75 mol L-1; temperature, 70 °C; 300 rpm.
these ODN molecules at the polymer-water interface.14 They also found that such ODN adsorbed onto positively charged particles as well as onto negatively charged ones, but in this latter case the small adsorbed amount strongly increased upon increasing ionic strength. HRF data were in favor of a flat conformation of the DNA fragments in the former case. A more complex situation occurred with the anionic latexes, depending upon the structure of the ODN, polymer surface coverage, and ionic strength. On the basis of these different results, several aspects need to be clarified concerning the adsorption behavior of ODN’s onto latex particles as a function of several pertinent parameters which were not fully taken into account in our previous studies. Firstly, the adsorption is examined in the case of another homopoly(nucleic acid) (poly(thymidylic acid)) using the same cationically charged latex particles as solid phase support and upon varying pH, ionic strength, the surface charge density of latex particles, and chain length of poly(thymidylic acid). Secondly, the adsorption behavior of this ODN is compared with that of other ODN’s such as dA30, dC35, and dT10C12G5, and a general discussion is focused onto the main features governing the adsorption of single-stranded DNA fragments onto polymer particles. II. Materials and Methods 1. Synthesis and Characterization of Poly(thymidylic acid) Oligodeoxyribonucleotides. The single-stranded oligodeoxyribonucleotides dT8, dT13, dT20, dT25, dT30, and dT35 bearing an aliphatic primary amine (H2N-(CH2)6-) at the 5′ terminus were synthesized by β-cyanoethyl phosphoramidite chemistry17 on a 1 µmol scale with an Applied Biosystems DNA/ RNA synthesizer (model 394). The monomethoxytrityl (MMT) amine protecting group was retained on the 5′ to simplify HPLC purification. All reagents and standard phosphoramidites are from Applied Biosystems (Foster City, CA) except N-MMTC6-amino-modifier phosphoramidite which is from Clontech Laboratories, Inc. (Palo Alto, CA). The cleavage of synthesized oligodeoxyribonucleotides from the solid support was carried out automatically on the synthesizer by 90 min of treatment with concentrated (30%) ammonium hydroxide solution. The full deprotection was then achieved by incubating the ammonia solution containing the cleaved oligodeoxyribonucleotide at 55 °C overnight. The crude oligodeoxyribonucleotides with 5′-MMT group were purified by reverse-phase HPLC using a Beckmann ODS (10 mm × 250 mm) column. Chromatography was performed at 7.7 mL/min with the following linear gradient: 0 min, 20% buffer A; 10 min, 30% buffer B; 30 min, 70% buffer B. Buffer A was 0.1 M triethylammonium acetate (TEAA, pH 7.0). Buffer B was 50% acetonitrile in 0.1 M TEAA (pH 7.0). Purified oligodeoxyribonucleotides were then detritylated with 2 mL of 80% acetic (17) Gait, M. J. Oligonucleotide Synthesis, a practical approach; IRL Press: Oxford, 1984.
acid for 1 h at room temperature and precipitated by addition of 3 M sodium acetate and cold ethanol (-20 °C). The purified products (98% purity) were quantified by measuring the UV absorption at 260 nm. The following equation was used
C)
[
]
A260nm × 100 0.92nT
(1)
where C is the concentration of the oligodeoxyribonucleotide (nmol/L), A260nm the absorption at 260 nm, and nT the number of thymidine nucleosides present in the poly(thymidylic acid). The molecular weight of dTn was Mw ) nT300 + 134 g mol-1 as calculated using a standard formula,18 where 134 and 300 are the molecular weights of the aliphatic primary amine group and the thymine nucleotides, respectively. 2. Preparation and Characterization of Aminated Latex Particles. Stable cationic latexes were prepared by either batch or shot-growth emulsifier-free emulsion polymerization of styrene and vinylbenzylamine hydrochloride (VBAH) using 2,2′-azobis(2-amidinopropane) dihydrochloride (V-50) as the initiator as described previously.19,20 The seed latex was obtained by batch emulsifier-free polymerization, according to a recipe given in Table 1. The shot-growth process in emulsion polymerization consisted of adding styrene, VBAH, and V-50 to the mixture after 80% conversion of the batch polymerization, in order to increase the surface charge density of latex particles. The polymerization was carried out in a 250 mL round-bottomed fournecked flask equipped with a glass anchor-shaped stirrer adjusted at 300 rpm, condenser, thermocouple, and nitrogen inlet. The temperature was checked to be constant at 70 °C. The recipe for seeded-batch polymerization is given in Table 2. In all cases, cleaning of latex particles was carried out by repetitive centrifugation (at least seven cycles). Latex particle diameter and distribution was determined by transmission electron microscopy (from Hitachi at CMABO in Claude Bernard University, Lyon I, France). As shown in Figure 1 for latex sample GAF11 and in Table 3 for all samples, latexes were found to be highly monodisperse with a uniformity ratio between 1.001 and 1.010. The surface charge density was determined by a colorimetric titration method20 elsewhere reported, and the results are given in Table 3. Only one latex (GAF11) was used for investigating the effect of various parameters (pH, ionic strength, zeta-potential, and oligodeoxyribonucleotide chain length) on the adsorption of oligodeoxyribonucleotides. The other latexes were used for studying the influence of the surface charge density on the ODNadsorbed amount. The zeta-potential (ζ) of the prepared latex particles were measured at 25 °C and at ionic strength 10-3 M NaCl, with Zetasizer III from Malvern Instruments, France. The electrophoretic mobilities measured were converted into zeta-potential (18) Brown, T.; Brown, D. J. In Oligodeoxyribonucleotides and analogues, A practical approch; Eckstein, S. F., Ed.; IRL Press: Oxford, 1991; pp 1-23. (19) Delair, T. French patent No. 9106068. (20) Ganachaud, F.; Mouterde, G.; Delair, T.; Elaı¨ssari, A.; Pichot, C. Polym. Adv. Tech. 1995, 6, 480.
Adsorption of Single-Stranded DNA Fragments
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Figure 2. Zeta-potential of aminated latex particles (GAF11) as a function of pH (25 °C, 10-3 M NaCl).
Figure 1. Transmission electron micrograph of styrene/VBAH copolymer latex particles (GAF11). Table 3. Polymerization Method and Surface Characteristics of Polymer Latexes latex code
Dn (nm)
Dw (nm)
Ua
σ (µC cm-2)b
polymerization method19
GAF2 GAF3 GAF1 GAF10 GAF11
473 190 118 218 241
473 192 119 219 243
1.001 1.01 1.004 1.002 1.005
18.95c 11.35 9.15 16.45 17.45
batch batch batch shot-growth shot-growth
a
The uniformity ratio is defined as U)Dw/Dn. b Surface charge density determined by a colorimetric titration method.20 c Only amidine surface groups onto the latex particles.
values using O’Brien and White’s theory21 according to the following equation
[
ζ˜ ln 2 (1 - exp(-zζ˜ ) 3ζ˜ 2 z µ˜ ) 2 κa -zζ˜ exp 2+ m 2
]
(2)
where µ˜ is the reduced electrophoretic mobility, a the particle’s radius, m related to the friction factor for small ions, z the valency of electrolyte, e the elementary charge, κ the reciprocal Debye length, ζ˜ ) (eζ)/kBT the reduced zeta-potential, kB the Boltzmann constant, and T the absolute temperature. Each point is the average of four measurements. 3. Adsorption Measurements. Oligodeoxyribonucleotide adsorption was calculated from the difference between the initial and the final amount of oligonucleotide in solution after incubation with a known latex surface area Σ (m2 g-1). A latex concentration of M (g) was used for the determination of adsorption isotherms. Samples of latex containing added oligodeoxyribonucleotide at a given initial concentration Ci (nmol mL-1) and buffer were shaken for 10 h at 25 °C and centrifuged for 10 min at 14 000 rpm. The ODN concentration in the supernatant Cf (nmol mL-1) was then determined by UV absorption using the HPLC system (at 260 nm wavelength). The amount of adsorbed ODN on latex surface (Ns) obtained by depletion measurements after 10 h was expressed in mg m-2. The following equation was used to determine the amount of ODN adsorbed Ns (mg m-2):
[
]
Ci - Cf MΣ
area, and M the mass of latex particles. Phosphate buffer was used to maintain the pH between 4.5 and 9.6. The ionic strength of each buffer was adjusted to a desired ionic strength by adding NaCl. To measure the rate of ODN adsorption onto latex particles at a given pH and ionic strength, the initial time of addition of ODN solution to each sample was noted with a stopwatch. The samples were shaken at a constant temperature. At different intervals of time, each sample was taken out of the shaker and filtrated on a 0.1 µm Millipore filter in order to measure the ODN concentration Cf in the serum.
III. Results and Discussion
( )
Ns ) v
Figure 3. Percent of adsorbed dT35 as a function of time (in minutes) at pH 5.0 and ionic strength 10-2 M for experiment performed with 2.24 nmol mL-1 dT35 solution and 1.9 mg mL-1 latex concentration (GAF11).
(3)
Here v is the volume of solution in mL, Σ the specific surface (21) Hunter, R. J. Zeta Potential in Colloid Science, Principles and Applications; Academic Press: London, 1981.
1. Zeta-Potential of Latex Particles. The zetapotential of latex sample (GAF11) as a function of pH is shown in Figure 2, from which it can be seen that the particles bearing positive charges originated from both the VBAH monomer (amine groups) and fragments of the cationic initiator residues (amidine groups). The zetapotential decreases as pH increases, reflecting that the isoelectric point (IEP) of the aminated latex particles12,13 is around pH 10. However, above the IEP the negative zeta-potential can be attributed to the presence of carboxylic groups originating from the hydrolysis of amidine groups. 2. Adsorption Kinetic of Poly(thymidylic acid) onto Cationic Latex Particles. A preliminary study was conducted on the kinetic behavior of the adsorption of poly(thymidylic acid) (dT35) onto one latex sample (GAF11) at pH 5.0 and 10-2 M ionic strength (NaCl). The adsorbed amount of dT35 versus time curve is reported in Figure 3, showing that adsorption equilibrium is almost completed within 20 min. The adsorption kinetic of ODN on the cationic latex particles was too fast to determine the adsorption rate at the beginning of the adsorption process using the static adsorption method. Figure 3 shows the adsorbed amount of ODN onto cationic latex particles as a funtion of time (at pH 5.0 and ionic strength 0.01M). 3. Adsorption Isotherms of Poly(thymidylic acid) onto Aminated Latex Particles. Adsorption isotherms
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Figure 4. Adsorption isotherms of dT35 onto aminated latex particles (GAF11) at 25 °C and ionic strength 10-2 M.
Figure 5. Maximal adsorbed amount of dT35 onto latex particles (GAF11) as a function of pH (25 °C, ionic strength 0.01 M).
at 10-2 ionic strength, in which the adsorbed amount Ns (mg m-2) is plotted versus the dT35 concentration in solution (Cf in mg mL-1), are shown in Figure 4 for three different pH values. All adsorption isotherms are of high affinity, and show well-defined plateaus. The adsorbed amount is found to decrease upon increasing the pH, as a result of the decrease of attractive interactions between ODN’s and the latex surface as shown in Figure 2. The adsorption isotherms look like a typical Langmuir type where the adsorbed amounts rapidly increase with the ODN concentration up to a plateau region. Since the bulk ODN concentration is almost nil in the initial part of the reported adsorption isotherms, it was not possible to express these data in terms of the Langmuir plot (1/Ns as a function of 1/Cf). 4. Effect of pH on the Maximum Adsorbed Amount of ODN onto Latex Particles. The adsorption of dT35 depends on the pH of the incubated medium as reported in Figure 5. By considering the effect of pH on the surface charge density of the latex particles, it seems that the adsorption of ODN onto latex particles decreases with decreasing the cationic surface charge density on the latex surface. The maximum adsorbed amount (1.0 mg m-2) at pH 4.0 is near the theoretical value of a close-packed monolayer of flat conformation, assuming that the ODN is in a cylindrical conformation of height L ) 35 × 3.4 Å and radius R ) 8 Å.22 According to previous investigations,12,13 electrostatic interactions were found to be the most important contribution upon increasing the net cationic charge on the latex surface. Comparing the results obtained for different ODN’s, one with three different nucleotides (dC12G5T10)12 and an other containing a fluorescein group FITC-dA30,13 we also found that the ODN adsorption onto the cationic latexes was dependent on the pH and the surface charge density of the latex, which confirms that electrostatic interactions are the driving forces. It must be noted that the maximal adsorbed amount of dC12G5T10 molecules12 (22) Cantor, C. R., Schimmel, P. R., Eds.; In Biophysical Chemistry; Freeman: New York, 1980; Vol. 3.
Ganachaud et al.
Figure 6. Maximal adsorbed amount of dT35 onto latex particles (GAF11) as a function of ionic strength and pH (at 25 °C).
(1.2 mg m-2) is close to that of dT35 (which is 1 mg m-2). This similarity in the maximal adsorbed amounts indicates that both ODN’s are adsorbed onto the cationic latex particles according to the same mechanism. The adsorbed amount of ODN at basic pH is still nonnegligible, reflecting that the adsorption is not only governed by electrostatic interactions. As already observed,13 it is worth mentioning that some ODN adsorption occurs in the basic pH region with a value in the same range (0.4 mg m-2) as in the case of negatively charged latex particles.12 This value is in the same range as that reported by Walker et al.15 for poly(thymidylic acid) of similar size. This could be ascribed to the contribution of nonelectrostatic forces (possibly hydrophobic interactions or hydrogen bonding) between the ODN and the particle surface. 5. Effect of Ionic Strength on the Maximum Adsorbed Amount of ODN onto Latex Particles. In order to better understand the adsorption mechanism of dT35 molecules on the cationic latex particles, the effect of ionic strength was investigated in the range 10-4-10-1 M NaCl. The results shown in Figure 6 suggest that ionic strength does not play a significant role in the adsorbed amount of oligodeoxyribonucleotide (negatively charged) on the latex particles (positively charged). The influence of ionic strength seems to have a more marked effect at pH 4 than at other pH’s. At acidic pH and for a low ionic strength (less than 10-2 M), the adsorbed amount increases as the electrolyte concentration increases and it reaches a plateau above 10-2 M. Such a result could suggest that part of the segments of dT35 are not involved in the attractive interactions with the cationic charges of the latex particles. Indeed, with a high level of adsorbed molecules, this would cause mutual repulsion between free segments; therefore increasing ionic strength would screen these repulsive forces, and more dT35 molecules could be adsorbed. Due to the difficulty to prepare a buffer (at pH’s 6, 7, 8) in the low salt concentration range ( 0.5) for a highly charged polyelectrolyte, the dielectric constant, κ the reciprocal Debye length, T the absolute temperature, kB the Boltzmann constant, and Ln(u) the nonelectrical free energy gain. The experimental values and the calculated ones are compared in Table 4. Quantitatively, the adsorbed amount increases with increasing surface charge density, and the calculated values are in fairly good agreement with the experimental ones as predicted using the Hesselink theory for various Ln(u) values. These results show that the nonelectrical free energy gain on the adsorption of ODN chain is between 1kBT and 10kBT within the experimental error on the ODN’s adsorbed amount ((0.1 mg m-2). IV. Conclusion In the present work, static aspects of the interactions between ODN’s and cationic latex particles have been investigated. At first, a detailed study was conducted on the adsorption behavior of a polythymidylic acid onto various amino-containing cationic polystyrene latexes. We focused on the influence of pertinent parameters (pH, ionic strength, surface charge density of the particles, length of the ODN) on the adsorption process. The following results may be emphasized: (i) Adsorption isotherms are generally of high-affinity type, especially at low pH, with an equilibrium state attained within 20 min. (ii) The adsorbed amount of ODN (Ns max) is strongly pHdependent, and the variation of Ns max can be similarly expressed in terms either of the surface charge density (σ) or of the corresponding zeta-potential of the bare latex particles. (iii) Ionic strength in the studied range does not play a significant role except in the low-pH region. (iv) The adsorbed amount of ODN is not dependent on the chain length of the ODN (if expressed in unit mass m-2 or base number m-2). From these results, it was concluded that the adsorption of polythymidylic acid is mostly governed by electrostatic interactions between the negative charges of the ODN and the cationic ones of the latex. This can be considered as a general trend concerning the adsorption behavior of homopoly(nucleic acid) and ODN’s onto cationic latexes, and in addition, there is no noticeable effect of the nature of the base on the maximal adsorbed amount. Moreover, the contribution of a nonionic term (hydrophobic forces or (29) Stigter, D. J. Phys. Chem. 1979, 83, 1670.
Adsorption of Single-Stranded DNA Fragments
hydrogen bonding) to the adsorption energy was confirmed and deduced by extrapolation of Ns at σ (or ζ) ) 0. The obtained adsorbed amount value was in the same range as that observed in the case of negatively charged latex particles.12 The prediction of the adsorbed quantities of ODN’s as a function of the cationic surface charge density was performed using the Hesselink approximate theory, assuming that the ODN can be treated as a small-size polyelectrolyte. This model suggests that in addition to electrostatic interactions (which are the driving forces in the adsorption phenomena) the nonionic interactions should be taken into account. Finally, the adsorption data obtained upon varying the chain length of the ODN could also provide information concerning the conformation of the adsorbed ODN onto the cationic latex particles. Due to the helical structure of the ODN’s,19 it might be considered that they are mostly adsorbed in a flat monolayer conformation, assuming that each ODN exhibits a cylindrical form. In addition, due to steric hindrance or the contribution of hydrophobic forces (or hydrogen bonding), some ODN’s might be loosely
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bound to the particles’ surface, with a part of the base sequence expanded out in the aqueous phase. This aspect is currently under investigation using neutron-scattering techniques with which more insight can be deduced as to whether the ODN is simply adsorbed or covalently attached through a terminal amino spacer. The results will be published later on. In conclusion, this study shows that the adsorption of single-stranded small DNA probes onto latex particles can be relatively well-described, provided a few parameters of the system are well-determined such as the nature and surface charge density of the latex particles, pH, ionic strength, and the ODN chain length. Acknowledgment. The authors are indebted to Dr. T. Delair (Bio-Me´rieux, Lyon, France) and to Dr. E. Pefferkorn (ICS-CRM-CNRS, Strasbourg, France) for helpful discussions and suggestions. T. Schmidt is also gratefully acknowledged for a careful reading of the English manuscript. LA960896E
