- Email: [email protected]
Hydrophilic magnetic latex for nucleic acid extraction, purification and concentration
Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
Hydrophilic magnetic latex for nucleic acid extraction, puri"cation and concentration A. ElamK ssari *, M. Rodrigue, F. Meunier , C. Herve UMR-2142 CNRS-bioMe& rieux, ENS de Lyon, 46 alle& e d+Italie, 69364 Lyon Cedex 07, France bioMe& rieux Laboratory, Parc d+activite& de Moulin a% Vent BaL t. 20, 33 rue du Dr Levy, 69200 Ve& nissieux, France
Abstract Core-shell magnetic latex particles bearing poly(N-isopropylacrylamide) in the shell were prepared by encapsulation of magnetic core using a precipitation polymerization process. The cationic character of the particles' surface is favorable for nucleic acid adsorption}desorption by controlling the pH and salinity of the medium. The concentration process of nucleic acids was presented and proven using DNA as a model. 2001 Elsevier Science B.V. All rights reserved. Keywords: Thermosensitive particles; Magnetic latex; Poly(N-isopropylacrylamide); Nucleic acids; Adsorption; Desorption; Core-shell particles
1. Introduction In recent years, an increasing interest has been devoted to the preparation of magnetic latex particles for diagnostic applications. The pioneer work in this "eld has been done by Ugelstad et al. [1], who reported the preparation of monosized magnetic microspheres of micrometer size. He also described their utilization as a particular support for biomolecules [2]. Several methodologies on the preparation of magnetic latexes have been investigated as reported by (i) Kondo et al [3] in the case of thermosensitive poly(styrene/N-isopropylacrylamide/methacrylic acid) magnetic particles, (ii) batch emulsion polymerization of styrene in the presence of magnetic iron oxides by Charmot et al.
* Corresponding author. Tel.: #33-4-72-72-83-64; fax: #334-72-72-85-33. E-mail address: [email protected] (A. ElamK ssari).
[4], (iii) heterocoagulation concept investigated by Furusawa et al. [5] and (iv) the recent method derived from Furusawa's approach by controlling di!erent steps involved in the preparation such as adsorption and encapsulation as reported by Sauzedde et al. [6]. It is worth mentioning that the target in the preparation of magnetic latexes is the elaboration of superparamagnetic magnetic latex particles with an appreciable amount of iron oxide, submicronic in size and with good size distribution. All elaborated magnetic latexes are principally used as a solid support for covalent immobilization of biomolecules involved in speci"c capture of a given target. In fact, the magnetic colloids are largely used in immunoassays (such as ELISA test) and nucleic acid "elds (ELOSA). The main objective in biomedical diagnosis is the elaboration of new magnetic latexes and convinced methodologies in order to enhance the sensitivity and the speci"city of the analysis. The problems encountered in
0304-8853/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 0 ) 0 1 2 4 0 - 3
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A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
this "eld and particularly that of nucleic acid probes is sensitivity. One possibility to partially resolve the behavior problem is to increase the concentration of the target (RNA and/or DNA) in the considered medium before speci"c capture and detection of the target probe. The introduction of appropriate magnetic latex particles for nucleic acid concentration is of interest. Therefore, the objective of this work is to report on the preparation and utilization of thermally sensitive hydrophilic magnetic latex particles for nucleic acid adsorption, concentration and ampli"cation.
2. Experimental 2.1. Preparation and characterization The thermosensitive magnetic latexes were prepared using the seed polymerization process. Boiled and deoxygenated water (40 ml) and 1 g of seed magnetic latex particles (EM1 100/20 from Prolabo) freshly cleaned were introduced into the polymerization reactor under a constant stream of nitrogen. 100 l of styrene was "rst added to the seed latex mixture while being stirred and proceed in a nitrogen atmosphere for 2 h at 703C. The initiator 2,2 -azobis(amidinopropane) dihydrochloride (0.01 g) was "rst added, then 0.325 g of Nisopropylacrylamide (NIPAM), 0.274 g of the crosslinker N,N-methylenebis(acrylamide) (MBA), 0.14 g of Triton X-405 and 0.4 g of functional monomer 2-aminoethylmethacrylamide hydrochloride (AEMH) were then added. The polymerizations were carried out during 18 h and the amount of functional monomer was varied in order to prepare latexes with various surface charge densities. Several magnetic latexes were chemically modi"ed by varying the polymerization recipe. In addition, a shot polymerization process was also performed in order to increase the cationic surface charge density of the magnetic latex particles.
average particle size as determined using transmission electron microscopy (TEM) and the surface charge density was chemically determined [7] with a range from 150 to 300 mol NH /g. 2.3. Adsorption protocol A given amount of thermally sensitive magnetic latex particles (100 l) was mixed with a nucleic acid solution in an Eppendorf tube. The volume was adjusted to 1 ml using phosphate bu!er. The mixture was incubated and slowly agitated for a set time and either at 203C or a given temperature. After magnetic separation of the magnetic latex particles, the free nucleic acid concentration was determined using the VIDAS automated system from bioMeH rieux [8] and the adsorbed amount of nucleic acids was determined from the di!erence between the initial and free nucleic acid concentrations. 2.4. Concentration and desorption protocols After the adsorption step, the magnetic latex particles bearing adsorbed nucleic acid molecules were isolated and redispersed in 100 l (bu!er solution at a given pH, ionic strength and at 203C). The desorbed amount of nucleic acid was then determined as described above for the adsorption step. 2.5. Bacterial strain The strain used in the study as a model was Staphylococcus epidermidis (ATCC 14990). Bacterial cultures were grown on agar (GTS medium, bioMeH rieux, France) or liquid medium (BrainHeart medium) and incubated at 373C. In some cases, Staphylococcus epidermidis was spiked in sputa in order to mimic one of the more di$cult clinical specimens to process.
2.2. Characterization
2.6. Polymerase chain reaction amplixcation
The colloidal characteristics of the prepared thermosensitive magnetic latexes are summarized as follows: 45% iron oxide content, 1 m was the
For isolates, a few grown colonies or amount of liquid culture were resuspended in sterile water. After checking by optical density, the concentration
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
of bacteria was adjusted by dilution in sterile water. Total nucleic acids were released from culture material by vortexing the bacterial suspension in the presence of glass beads. A 20 l aliquot of the DNA to be studied (raw or puri"ed specimen) was added directly to the PCR. Alternatively, PCR reagents are added directly to the magnetic latex particles when elution is omitted. The 16S hyper-variable region was PCR ampli"ed with speci"c primers Spi1 (position 1013}1034) and Spi4 (position 1598}1683). PCR ampli"cation was carried out in a 100 l reaction volume containing 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl , 5% (vol/vol) dimethyl sulfoxide, 15 M (each) primer, 200 M (each) deoxynucleotide triphosphates, and 1.5U of Taq polymerase (Ampli Taq, Perkin-Elmer). PCR was performed in a Perkin-Elmer 2400 thermal cycler with an initial denaturation step at 943C for 3 min, 653C for 1 min and cycling conditions of 723C for 1 min, 943C for 1 min and 653C for 2 min for 35 cycles and 723C for 5 min for the last cycle. 2.7. Amplicons detection PCR products were analyzed by agarose gel electrophoresis (presence or absence of a band of the expected size) or Vidas analysis [9]. In short, amplicons are placed into the sample well of the 10-well polypropylene disposable strip of the Vidas automated system (bioMeH rieux, Marcy, France). This strip already contained all necessary reagents for the subsequent hybridization steps in separate wells: labeled probe, washing bu!er and substrate. The strip was processed automatically in the Vidas system. Two DNA oligonucleotides speci"c for Staphylococcus epidermidis were used in a sandwich hybridization format as described by Allibert et al. [10]. One oligonucleotide was coated inside the solid surface of the pipette tip, the solid-phase receptacle (SPR) and acted as a capture probe. A second oligonucleotide was modi"ed by introducing alkaline phosphatase and used as a detection probe. Upon completion of the assay, the alkaline phosphatase substrate, 4-methylumbelliferyl phosphate (0.6 mM), was pumped from the
129
"nal well, an optically clear cuvette. The alkaline phosphatase from the SPR sandwich catalyses its conversion into a #uorescent product, 4-methylumbelliferone. The #uorescence intensity (450 nm) of the cuvette content was measured by the Vidas optical scanner before and after contact with the SPR. Results were analyzed automatically and expressed in relative #uorescence value (RFV). Test values less than 200 RFV were considered as being negative results while test values greater than or equal to 200 RFV were considered to be positive. The capture and detection probes were Spi5: position1051}1071 (capture probe) and Spi6: position 1028}1048 (detection probe), respectively. 2.8. Particle binding and elution Specimen 100 l was added to 900 l of phosphate bu!er (NaH PO , 10 mM, pH 4.68) contain ing 200 g of magnetic latex particles. After an incubation time of 30 min at 203C, the sample was submitted to a 5 min separation step using a magnet. Supernatant was discarded and elution, when performed, was realized by adding 100 l of elution bu!er (50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl , 5%(vol/vol) dimethyl sulfoxide). This elution bu!er was, in this case, the PCR bu!er but can be any other as long as it is taken into account that a basic pH and high salt concentration are needed.
3. Results and discussions 3.1. Adsorption kinetics of nucleic acids The adsorption kinetics of RNA molecules onto the hydrophilic cationic magnetic latexes was "rst investigated and the results obtained are reported in Fig. 1. The adsorbed amount was found to increase as the incubation time increased up to 10 min, which explains the rapid adsorption process. Then, after 15 min, the adsorption reaches a plateau leading to an adsorbed amount of 4 mg/g under investigation conditions (pH&5.2, 10\ M NaCl, at 203C). The observed behavior re#ects that the adsorption of such polyelectrolytes is rapid as was already been observed in the case of
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Fig. 1. Adsorbed amount of nucleic acids as a function of time at pH&5.2, 100 g of latex particles, 2.5 g RNA, 10\ ionic strength at 203C.
single-stranded DNA(ssDNA) fragments (oligonucleotides) adsorption onto cationic polystyrene latex particles as reported by ElamK ssari et al. [11}14] and for RNA adsorption onto cationic hydrogel particles [15]. The e!ect of temperature on the maximum adsorbed amount was also investigated. Surprisingly, no drastic e!ect of temperature was found, which explains that the adsorption of RNA is principally governed by electrostatic interactions. 3.2. Ewect of pH on the adsorption of nucleic acids The adsorption of the nucleic acids was investigated at 203C, 10\ M NaCl and 30 min as the incubation time. The initial nucleic acid concentration and incubation time were selected according to the adsorption kinetics discussed above. As expected, the adsorbed amount of nucleic acids decreases when pH of the incubation medium is increased. In fact, the increases in the pH leads principally to a!ecting the concentration of the charges involved in the interaction process between nucleic acids negatively charged and cationic charges of the magnetic latex particles. Similar behavior has been already reported for small ssDNA fragments adsorption onto cationic polystyrene latexes bearing both amidine and amine groups [11}14] and for RNA adsorption onto cationic hydrogel particles [15]. The attractive electrostatic interactions were found to be the driving forces in the adsorption process (Fig. 2).
Fig. 2. The e!ect of pH on the maximal adsorbed amount of RNA molecules onto magnetic latex particles with 100 g of latex particles, 1 g RNA, 10\ ionic strength, incubation time 30 min at 203C.
3.3. Ewect of ionic strength on the adsorption of nucleic acids The in#uence of ionic strength on the nucleic acid adsorbed amount was studied using phosphate bu!er at a given salt concentration, pH&5.2 and at 203C. The adsorbed amount is found to decrease as the ionic strength of the incubation medium increases, as a result of the decrease in attractive interactions between nucleic acid molecules and hydrophilic cationic latex surface as shown in Fig. 3. The e!ect of salt on the adsorbed amount of nucleic acids can be attributed to two major e!ects: (i) The increase in the ionic strength leads principally to reduce the attractive electrostatic interactions between nucleic acids negatively charged and latex particles bearing cationic groups. Consequently, the adsorbed amount was reduced, and (ii) the salinity drastically in#uences the solvency of the thermally sensitive polymers, and in this study, the poly(NIPAM) shell. In fact, increasing the electrolyte concentration leads to increasing the Flory}Huggins [16] interactions parameter between the polymer and water, then resulting in the reduction of the poly(NIPAM) solvency. 3.4. Ewect of surface charge density The e!ect of surface charge density on the adsorbed amount of nucleic acid molecules can be
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
Fig. 3. The e!ect of ionic strength on the RNA adsorbed amount with 100 g of latex particles, 2.5 g RNA, incubation time 30 min at 203C at pH"5.2.
deduced from the pH e!ect. In fact, the pH a!ects drastically the protonated groups on the particles leading to a high amount adsorbed at acidic pH. A good way to increase the amount adsorbed is to operate using a highly positively charged magnetic particle at acidic pH. To point out the e!ect of charge density on the adsorbed amount of RNA, several latexes bearing increasing surface charge density were prepared. As expected, the adsorbed amount of RNA increases when the positive charges on the latex particles are increased. 3.5. Ewect of pH on the nucleic acids desorbed amount The desorption of preadsorbed nucleic acid molecules was investigated as a function of pH of the desorption medium. As expected, the desorbed amount increases as the pH of the incubation medium is increased. This behavior can be attributed to the reduction of the surface charge induced by the pH of the medium. As a result, the reduction in the attractive charges on the latex particles leads to an increase in the desorbed amount as can be seen in Fig. 4. Moreover, the e!ect of pH on the desorption behavior supported the theory that the adsorption}desorption process of such highly charged polyelectrolytes is principally governed by charge}charge electrostatic interactions onto such hydrophilic and cationic latexes.
131
Fig. 4. The e!ect of pH on the desorption of preadsorbed nucleic acid molecules onto cationic magnetic latex particles. The adsorption was performed using 10 mM phosphate bu!er, pH&5.2, 10\ M NaCl at 203C during 180 min incubation time.
3.6. Ewect of ionic strength on the nucleic acids desorbed amount Depending on the in#uence of ionic strength on the adsorption process of nucleic acids, the desorption will be also a!ected. In fact, the desorption of preadsorbed nucleic acid molecules was drastically altered by the salinity of the desorption medium (see Fig. 5). The behavior observed can be attributed to the combination of two pertinent parameters: (i) salinity leads principally to reduce the magnitude of the attractive forces and (ii) increases in the salinity induced a shrinkage of the poly(NIPAM) shell, consequently, the desorption was increased according to low accessibility of the cationic binding sites and also increases in the steric hindrance of the interfacial nucleic acid molecules. Similar result tendencies have been also reported by ElamK ssari et al. [15] by studying the adsorption of RNA onto hydrophilic thermosensitive latex particles. Moreover, high salinity favors the desorption of preadsorbed nucleic acid molecules. 3.7. Amplixcation of nucleic acids The ampli"cation of the captured target was investigated in both cases (i) direct ampli"cation of the adsorbed target on the latex particles and (ii) after desorption in a small volume bu!er solution (i.e. concentration of the target and removal of the magnetic latex particles).
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
132
Table 2 Sensitivity study of latex particles. Vidas analysis of amplicons after an elution step. The results are expressed in relative #uorescence value ("RFV) Number of bacteria per assay 0 40
Fig. 5. E!ect of added salt (NaCl) concentration on desorption of nucleic acids from the magnetic latex particles at pH 9.2 and 203C. The adsorbed amount of nucleic acid at pH&5.2 and 10\ M NaCl was 4 mg/g.
10 76
10 60
10 662
10 4651
10 9264
10 10 457
Table 3 Vidas results (RFV value) obtained with bacteria spiked in water or sediment and with or without puri"cation on magnetic latex particles. As an example `Sputum 1 non-puri"eda showed the inhibitory e!ect of this kind of samples for the PCR Number of bacteria per assay
Table 1 E!ect of latex amount in PCR reaction Amount of particles per assay Negative Positive control control ! #
5 ng
50 ng 500 ng 5 g
50 g 500 g
#
#
!
#
#
!
PCR was performed directly on beads. A positive result means the presence of a bead of the expected size by agarose electrophoresis.
Bacteria in H O Sputum 1 non-puri"ed Sputum 1 puri"ed Sputum 2 puri"ed Sputum 3 puri"ed
0
10
10
10
10
10
NT
NT
1152 4201
10
NT
11
25
NT
40 28
9
10 5898
10
23
663
2911 5076
7350
98
160
306 2612
9327
NT
NT
720 5722
10771
NT: not tested. Puri"ed"after adsoprtion using magnetic particles.
Nucleic acids released from 10 bacteria were captured with di!erent amounts of latex particles; ampli"cation was performed directly on particles. A negative (water) and positive control (ampli"cation of 10 free nucleic acids) were tested in parallel. Ten microliters of each was analyzed on agarose gel. The results obtained are summarized in Table 1. No more than 5 g of particles can be incorporated in PCR without inhibition, probably coming from particles. Such an amount (5 g) of particles is insu$cient to capture all nucleic acids (see above) usually encountered in di!erent clinical samples. Then, 200 g of latex particles was tested and found to be in the lower limits of providing the capture capacity required. For such an amount, an elution step is necessary to perform PCR. Concerning other ampli"cation technologies (LCR, SDA, NASBA, TMA, etc.), tests should be done to check inhibitory e!ects.
Ampli"cation of the adsorbed target after elution from the latex particles was performed by "rst lysing di!erent amounts of bacteria (10}10 per assay and then releasing absorbed nucleic acids and concentrating them on 200 g of magnetic latex particles. Then, the PCR was performed after an elution step and 50 l of PCR product were analyzed with the Vidas assay (Table 2). Ten thousand bacteria were observed as the lower limit of sensitivity. This result, even if encouraging, could probably be increased by optimizing the lysis, adsorption, desorption steps, as well as the detection step. Sputum is one of the most di$cult clinical specimens to work with and is known to be quite inhibitory. However, in order to assess the e$ciency of latex particles in purifying nucleic acids, we spiked sputum sediments with di!erent amounts of
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
bacteria. After a lysis step, these specimens were PCR ampli"ed with or without puri"cation; then, after an elution step, 50 l of amplicons were analyzed using the Vidas assay (Table 3). We demonstrated the e$ciency of the latex particles in removing inhibitors coming from clinical specimens. Sensitivity is in accordance with the previous study ranging from 10 to 10 bacteria per assay. These very encouraging results are and level of sensitivity could probably be increased by optimizing all steps involved in the process.
4. Conclusion The adsorption and desorption of nucleic acid molecules can by controlled by monitoring the interaction between the prepared magnetic latex particles and the nucleic acids considered. The adsorption process of nucleic acids onto such cationic hydrophilic and magnetic core-shell latex particles was found to be governed by attractive electrostatic interaction as expected. In fact, the adsorption was favored principally at acidic pH and low salinity. The desorption study pointed out that desorption is not complete under the investigated conditions such as basic pH and high salinity. In fact, the desorbed amount was found to be lower than 90% of the initial adsorbed concentration. Moreover, even if the desorbed amount is lower than 100%, the desorption in low "nal volumes leads to a drastic increase of the nucleic acid concentration in the desired medium to ensure increased sensitivity of the diagnostic test.
133
The e$ciency of the magnetic latex particles to concentrate nucleic acids was demonstrated both with synthetic and biological targets. In addition, the magnetic particles were, also, e$cient in removing inhibitors from clinical specimens. As this was a feasibility study, these encouraging results can be further optimized along di!erent steps of the process. Experiments with RNA targets are under way where, depending on the ampli"cation technology used, the elution step can be omitted. In conclusion, magnetic latex particles are, thus, useful as puri"cation and concentration tools in a sample preparation step before ampli"cation technologies in the diagnostic "eld.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
J. Ugelstad et al., Prog. Polym. Sci. 17 (1992) 87. J. Ugelstad et al., Polym. Int. 30 (1993) 157. A. Kondo et al., J. Ferment. Bioeng. 84 (1997) 337. D. Charmot, Prog. Colloid Polym. Sci. 76 (1989) 94. K. Furusawa et al., Colloid Polym. Sci. 272 (1994) 1104. F. Sauzedde et al., Colloid Polym. Sci. 277 (1999) 846. F. Ganachaud et al., Polym. Adv. Technol. 6 (1995) 480. M.N. Erout et al., Bioconjugate Chem. 7 (1996) 568. C. Mabilat et al., Int. J. Food Microbiol. 28 (1996) 333. P. Allibert et al., Eur. J. Biomed. Technol. 14 (1992) 152. A. Elaissari et al., A. Colloids Surf. 83 (1994) 25. A. Elaissari et al., Langmuir 11 (1995) 1261. F. Ganachaud et al., Langmuir 13 (1997) 7021. F. Ganachaud et al., Langmuir 13 (1997) 701. A. Elaissari et al., J. Biomater. Sci. Polym. Edn. 10 (1999) 403. P.G. de Gennes, Cornell University Press, Ithaca, 1979.
Hydrophilic magnetic latex for nucleic acid extraction, puri"cation and concentration A. ElamK ssari *, M. Rodrigue, F. Meunier , C. Herve UMR-2142 CNRS-bioMe& rieux, ENS de Lyon, 46 alle& e d+Italie, 69364 Lyon Cedex 07, France bioMe& rieux Laboratory, Parc d+activite& de Moulin a% Vent BaL t. 20, 33 rue du Dr Levy, 69200 Ve& nissieux, France
Abstract Core-shell magnetic latex particles bearing poly(N-isopropylacrylamide) in the shell were prepared by encapsulation of magnetic core using a precipitation polymerization process. The cationic character of the particles' surface is favorable for nucleic acid adsorption}desorption by controlling the pH and salinity of the medium. The concentration process of nucleic acids was presented and proven using DNA as a model. 2001 Elsevier Science B.V. All rights reserved. Keywords: Thermosensitive particles; Magnetic latex; Poly(N-isopropylacrylamide); Nucleic acids; Adsorption; Desorption; Core-shell particles
1. Introduction In recent years, an increasing interest has been devoted to the preparation of magnetic latex particles for diagnostic applications. The pioneer work in this "eld has been done by Ugelstad et al. [1], who reported the preparation of monosized magnetic microspheres of micrometer size. He also described their utilization as a particular support for biomolecules [2]. Several methodologies on the preparation of magnetic latexes have been investigated as reported by (i) Kondo et al [3] in the case of thermosensitive poly(styrene/N-isopropylacrylamide/methacrylic acid) magnetic particles, (ii) batch emulsion polymerization of styrene in the presence of magnetic iron oxides by Charmot et al.
* Corresponding author. Tel.: #33-4-72-72-83-64; fax: #334-72-72-85-33. E-mail address: [email protected] (A. ElamK ssari).
[4], (iii) heterocoagulation concept investigated by Furusawa et al. [5] and (iv) the recent method derived from Furusawa's approach by controlling di!erent steps involved in the preparation such as adsorption and encapsulation as reported by Sauzedde et al. [6]. It is worth mentioning that the target in the preparation of magnetic latexes is the elaboration of superparamagnetic magnetic latex particles with an appreciable amount of iron oxide, submicronic in size and with good size distribution. All elaborated magnetic latexes are principally used as a solid support for covalent immobilization of biomolecules involved in speci"c capture of a given target. In fact, the magnetic colloids are largely used in immunoassays (such as ELISA test) and nucleic acid "elds (ELOSA). The main objective in biomedical diagnosis is the elaboration of new magnetic latexes and convinced methodologies in order to enhance the sensitivity and the speci"city of the analysis. The problems encountered in
0304-8853/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 0 ) 0 1 2 4 0 - 3
128
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
this "eld and particularly that of nucleic acid probes is sensitivity. One possibility to partially resolve the behavior problem is to increase the concentration of the target (RNA and/or DNA) in the considered medium before speci"c capture and detection of the target probe. The introduction of appropriate magnetic latex particles for nucleic acid concentration is of interest. Therefore, the objective of this work is to report on the preparation and utilization of thermally sensitive hydrophilic magnetic latex particles for nucleic acid adsorption, concentration and ampli"cation.
2. Experimental 2.1. Preparation and characterization The thermosensitive magnetic latexes were prepared using the seed polymerization process. Boiled and deoxygenated water (40 ml) and 1 g of seed magnetic latex particles (EM1 100/20 from Prolabo) freshly cleaned were introduced into the polymerization reactor under a constant stream of nitrogen. 100 l of styrene was "rst added to the seed latex mixture while being stirred and proceed in a nitrogen atmosphere for 2 h at 703C. The initiator 2,2 -azobis(amidinopropane) dihydrochloride (0.01 g) was "rst added, then 0.325 g of Nisopropylacrylamide (NIPAM), 0.274 g of the crosslinker N,N-methylenebis(acrylamide) (MBA), 0.14 g of Triton X-405 and 0.4 g of functional monomer 2-aminoethylmethacrylamide hydrochloride (AEMH) were then added. The polymerizations were carried out during 18 h and the amount of functional monomer was varied in order to prepare latexes with various surface charge densities. Several magnetic latexes were chemically modi"ed by varying the polymerization recipe. In addition, a shot polymerization process was also performed in order to increase the cationic surface charge density of the magnetic latex particles.
average particle size as determined using transmission electron microscopy (TEM) and the surface charge density was chemically determined [7] with a range from 150 to 300 mol NH /g. 2.3. Adsorption protocol A given amount of thermally sensitive magnetic latex particles (100 l) was mixed with a nucleic acid solution in an Eppendorf tube. The volume was adjusted to 1 ml using phosphate bu!er. The mixture was incubated and slowly agitated for a set time and either at 203C or a given temperature. After magnetic separation of the magnetic latex particles, the free nucleic acid concentration was determined using the VIDAS automated system from bioMeH rieux [8] and the adsorbed amount of nucleic acids was determined from the di!erence between the initial and free nucleic acid concentrations. 2.4. Concentration and desorption protocols After the adsorption step, the magnetic latex particles bearing adsorbed nucleic acid molecules were isolated and redispersed in 100 l (bu!er solution at a given pH, ionic strength and at 203C). The desorbed amount of nucleic acid was then determined as described above for the adsorption step. 2.5. Bacterial strain The strain used in the study as a model was Staphylococcus epidermidis (ATCC 14990). Bacterial cultures were grown on agar (GTS medium, bioMeH rieux, France) or liquid medium (BrainHeart medium) and incubated at 373C. In some cases, Staphylococcus epidermidis was spiked in sputa in order to mimic one of the more di$cult clinical specimens to process.
2.2. Characterization
2.6. Polymerase chain reaction amplixcation
The colloidal characteristics of the prepared thermosensitive magnetic latexes are summarized as follows: 45% iron oxide content, 1 m was the
For isolates, a few grown colonies or amount of liquid culture were resuspended in sterile water. After checking by optical density, the concentration
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
of bacteria was adjusted by dilution in sterile water. Total nucleic acids were released from culture material by vortexing the bacterial suspension in the presence of glass beads. A 20 l aliquot of the DNA to be studied (raw or puri"ed specimen) was added directly to the PCR. Alternatively, PCR reagents are added directly to the magnetic latex particles when elution is omitted. The 16S hyper-variable region was PCR ampli"ed with speci"c primers Spi1 (position 1013}1034) and Spi4 (position 1598}1683). PCR ampli"cation was carried out in a 100 l reaction volume containing 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl , 5% (vol/vol) dimethyl sulfoxide, 15 M (each) primer, 200 M (each) deoxynucleotide triphosphates, and 1.5U of Taq polymerase (Ampli Taq, Perkin-Elmer). PCR was performed in a Perkin-Elmer 2400 thermal cycler with an initial denaturation step at 943C for 3 min, 653C for 1 min and cycling conditions of 723C for 1 min, 943C for 1 min and 653C for 2 min for 35 cycles and 723C for 5 min for the last cycle. 2.7. Amplicons detection PCR products were analyzed by agarose gel electrophoresis (presence or absence of a band of the expected size) or Vidas analysis [9]. In short, amplicons are placed into the sample well of the 10-well polypropylene disposable strip of the Vidas automated system (bioMeH rieux, Marcy, France). This strip already contained all necessary reagents for the subsequent hybridization steps in separate wells: labeled probe, washing bu!er and substrate. The strip was processed automatically in the Vidas system. Two DNA oligonucleotides speci"c for Staphylococcus epidermidis were used in a sandwich hybridization format as described by Allibert et al. [10]. One oligonucleotide was coated inside the solid surface of the pipette tip, the solid-phase receptacle (SPR) and acted as a capture probe. A second oligonucleotide was modi"ed by introducing alkaline phosphatase and used as a detection probe. Upon completion of the assay, the alkaline phosphatase substrate, 4-methylumbelliferyl phosphate (0.6 mM), was pumped from the
129
"nal well, an optically clear cuvette. The alkaline phosphatase from the SPR sandwich catalyses its conversion into a #uorescent product, 4-methylumbelliferone. The #uorescence intensity (450 nm) of the cuvette content was measured by the Vidas optical scanner before and after contact with the SPR. Results were analyzed automatically and expressed in relative #uorescence value (RFV). Test values less than 200 RFV were considered as being negative results while test values greater than or equal to 200 RFV were considered to be positive. The capture and detection probes were Spi5: position1051}1071 (capture probe) and Spi6: position 1028}1048 (detection probe), respectively. 2.8. Particle binding and elution Specimen 100 l was added to 900 l of phosphate bu!er (NaH PO , 10 mM, pH 4.68) contain ing 200 g of magnetic latex particles. After an incubation time of 30 min at 203C, the sample was submitted to a 5 min separation step using a magnet. Supernatant was discarded and elution, when performed, was realized by adding 100 l of elution bu!er (50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl , 5%(vol/vol) dimethyl sulfoxide). This elution bu!er was, in this case, the PCR bu!er but can be any other as long as it is taken into account that a basic pH and high salt concentration are needed.
3. Results and discussions 3.1. Adsorption kinetics of nucleic acids The adsorption kinetics of RNA molecules onto the hydrophilic cationic magnetic latexes was "rst investigated and the results obtained are reported in Fig. 1. The adsorbed amount was found to increase as the incubation time increased up to 10 min, which explains the rapid adsorption process. Then, after 15 min, the adsorption reaches a plateau leading to an adsorbed amount of 4 mg/g under investigation conditions (pH&5.2, 10\ M NaCl, at 203C). The observed behavior re#ects that the adsorption of such polyelectrolytes is rapid as was already been observed in the case of
130
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
Fig. 1. Adsorbed amount of nucleic acids as a function of time at pH&5.2, 100 g of latex particles, 2.5 g RNA, 10\ ionic strength at 203C.
single-stranded DNA(ssDNA) fragments (oligonucleotides) adsorption onto cationic polystyrene latex particles as reported by ElamK ssari et al. [11}14] and for RNA adsorption onto cationic hydrogel particles [15]. The e!ect of temperature on the maximum adsorbed amount was also investigated. Surprisingly, no drastic e!ect of temperature was found, which explains that the adsorption of RNA is principally governed by electrostatic interactions. 3.2. Ewect of pH on the adsorption of nucleic acids The adsorption of the nucleic acids was investigated at 203C, 10\ M NaCl and 30 min as the incubation time. The initial nucleic acid concentration and incubation time were selected according to the adsorption kinetics discussed above. As expected, the adsorbed amount of nucleic acids decreases when pH of the incubation medium is increased. In fact, the increases in the pH leads principally to a!ecting the concentration of the charges involved in the interaction process between nucleic acids negatively charged and cationic charges of the magnetic latex particles. Similar behavior has been already reported for small ssDNA fragments adsorption onto cationic polystyrene latexes bearing both amidine and amine groups [11}14] and for RNA adsorption onto cationic hydrogel particles [15]. The attractive electrostatic interactions were found to be the driving forces in the adsorption process (Fig. 2).
Fig. 2. The e!ect of pH on the maximal adsorbed amount of RNA molecules onto magnetic latex particles with 100 g of latex particles, 1 g RNA, 10\ ionic strength, incubation time 30 min at 203C.
3.3. Ewect of ionic strength on the adsorption of nucleic acids The in#uence of ionic strength on the nucleic acid adsorbed amount was studied using phosphate bu!er at a given salt concentration, pH&5.2 and at 203C. The adsorbed amount is found to decrease as the ionic strength of the incubation medium increases, as a result of the decrease in attractive interactions between nucleic acid molecules and hydrophilic cationic latex surface as shown in Fig. 3. The e!ect of salt on the adsorbed amount of nucleic acids can be attributed to two major e!ects: (i) The increase in the ionic strength leads principally to reduce the attractive electrostatic interactions between nucleic acids negatively charged and latex particles bearing cationic groups. Consequently, the adsorbed amount was reduced, and (ii) the salinity drastically in#uences the solvency of the thermally sensitive polymers, and in this study, the poly(NIPAM) shell. In fact, increasing the electrolyte concentration leads to increasing the Flory}Huggins [16] interactions parameter between the polymer and water, then resulting in the reduction of the poly(NIPAM) solvency. 3.4. Ewect of surface charge density The e!ect of surface charge density on the adsorbed amount of nucleic acid molecules can be
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
Fig. 3. The e!ect of ionic strength on the RNA adsorbed amount with 100 g of latex particles, 2.5 g RNA, incubation time 30 min at 203C at pH"5.2.
deduced from the pH e!ect. In fact, the pH a!ects drastically the protonated groups on the particles leading to a high amount adsorbed at acidic pH. A good way to increase the amount adsorbed is to operate using a highly positively charged magnetic particle at acidic pH. To point out the e!ect of charge density on the adsorbed amount of RNA, several latexes bearing increasing surface charge density were prepared. As expected, the adsorbed amount of RNA increases when the positive charges on the latex particles are increased. 3.5. Ewect of pH on the nucleic acids desorbed amount The desorption of preadsorbed nucleic acid molecules was investigated as a function of pH of the desorption medium. As expected, the desorbed amount increases as the pH of the incubation medium is increased. This behavior can be attributed to the reduction of the surface charge induced by the pH of the medium. As a result, the reduction in the attractive charges on the latex particles leads to an increase in the desorbed amount as can be seen in Fig. 4. Moreover, the e!ect of pH on the desorption behavior supported the theory that the adsorption}desorption process of such highly charged polyelectrolytes is principally governed by charge}charge electrostatic interactions onto such hydrophilic and cationic latexes.
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Fig. 4. The e!ect of pH on the desorption of preadsorbed nucleic acid molecules onto cationic magnetic latex particles. The adsorption was performed using 10 mM phosphate bu!er, pH&5.2, 10\ M NaCl at 203C during 180 min incubation time.
3.6. Ewect of ionic strength on the nucleic acids desorbed amount Depending on the in#uence of ionic strength on the adsorption process of nucleic acids, the desorption will be also a!ected. In fact, the desorption of preadsorbed nucleic acid molecules was drastically altered by the salinity of the desorption medium (see Fig. 5). The behavior observed can be attributed to the combination of two pertinent parameters: (i) salinity leads principally to reduce the magnitude of the attractive forces and (ii) increases in the salinity induced a shrinkage of the poly(NIPAM) shell, consequently, the desorption was increased according to low accessibility of the cationic binding sites and also increases in the steric hindrance of the interfacial nucleic acid molecules. Similar result tendencies have been also reported by ElamK ssari et al. [15] by studying the adsorption of RNA onto hydrophilic thermosensitive latex particles. Moreover, high salinity favors the desorption of preadsorbed nucleic acid molecules. 3.7. Amplixcation of nucleic acids The ampli"cation of the captured target was investigated in both cases (i) direct ampli"cation of the adsorbed target on the latex particles and (ii) after desorption in a small volume bu!er solution (i.e. concentration of the target and removal of the magnetic latex particles).
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Table 2 Sensitivity study of latex particles. Vidas analysis of amplicons after an elution step. The results are expressed in relative #uorescence value ("RFV) Number of bacteria per assay 0 40
Fig. 5. E!ect of added salt (NaCl) concentration on desorption of nucleic acids from the magnetic latex particles at pH 9.2 and 203C. The adsorbed amount of nucleic acid at pH&5.2 and 10\ M NaCl was 4 mg/g.
10 76
10 60
10 662
10 4651
10 9264
10 10 457
Table 3 Vidas results (RFV value) obtained with bacteria spiked in water or sediment and with or without puri"cation on magnetic latex particles. As an example `Sputum 1 non-puri"eda showed the inhibitory e!ect of this kind of samples for the PCR Number of bacteria per assay
Table 1 E!ect of latex amount in PCR reaction Amount of particles per assay Negative Positive control control ! #
5 ng
50 ng 500 ng 5 g
50 g 500 g
#
#
!
#
#
!
PCR was performed directly on beads. A positive result means the presence of a bead of the expected size by agarose electrophoresis.
Bacteria in H O Sputum 1 non-puri"ed Sputum 1 puri"ed Sputum 2 puri"ed Sputum 3 puri"ed
0
10
10
10
10
10
NT
NT
1152 4201
10
NT
11
25
NT
40 28
9
10 5898
10
23
663
2911 5076
7350
98
160
306 2612
9327
NT
NT
720 5722
10771
NT: not tested. Puri"ed"after adsoprtion using magnetic particles.
Nucleic acids released from 10 bacteria were captured with di!erent amounts of latex particles; ampli"cation was performed directly on particles. A negative (water) and positive control (ampli"cation of 10 free nucleic acids) were tested in parallel. Ten microliters of each was analyzed on agarose gel. The results obtained are summarized in Table 1. No more than 5 g of particles can be incorporated in PCR without inhibition, probably coming from particles. Such an amount (5 g) of particles is insu$cient to capture all nucleic acids (see above) usually encountered in di!erent clinical samples. Then, 200 g of latex particles was tested and found to be in the lower limits of providing the capture capacity required. For such an amount, an elution step is necessary to perform PCR. Concerning other ampli"cation technologies (LCR, SDA, NASBA, TMA, etc.), tests should be done to check inhibitory e!ects.
Ampli"cation of the adsorbed target after elution from the latex particles was performed by "rst lysing di!erent amounts of bacteria (10}10 per assay and then releasing absorbed nucleic acids and concentrating them on 200 g of magnetic latex particles. Then, the PCR was performed after an elution step and 50 l of PCR product were analyzed with the Vidas assay (Table 2). Ten thousand bacteria were observed as the lower limit of sensitivity. This result, even if encouraging, could probably be increased by optimizing the lysis, adsorption, desorption steps, as well as the detection step. Sputum is one of the most di$cult clinical specimens to work with and is known to be quite inhibitory. However, in order to assess the e$ciency of latex particles in purifying nucleic acids, we spiked sputum sediments with di!erent amounts of
A. Elan( ssari et al. / Journal of Magnetism and Magnetic Materials 225 (2001) 127}133
bacteria. After a lysis step, these specimens were PCR ampli"ed with or without puri"cation; then, after an elution step, 50 l of amplicons were analyzed using the Vidas assay (Table 3). We demonstrated the e$ciency of the latex particles in removing inhibitors coming from clinical specimens. Sensitivity is in accordance with the previous study ranging from 10 to 10 bacteria per assay. These very encouraging results are and level of sensitivity could probably be increased by optimizing all steps involved in the process.
4. Conclusion The adsorption and desorption of nucleic acid molecules can by controlled by monitoring the interaction between the prepared magnetic latex particles and the nucleic acids considered. The adsorption process of nucleic acids onto such cationic hydrophilic and magnetic core-shell latex particles was found to be governed by attractive electrostatic interaction as expected. In fact, the adsorption was favored principally at acidic pH and low salinity. The desorption study pointed out that desorption is not complete under the investigated conditions such as basic pH and high salinity. In fact, the desorbed amount was found to be lower than 90% of the initial adsorbed concentration. Moreover, even if the desorbed amount is lower than 100%, the desorption in low "nal volumes leads to a drastic increase of the nucleic acid concentration in the desired medium to ensure increased sensitivity of the diagnostic test.
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The e$ciency of the magnetic latex particles to concentrate nucleic acids was demonstrated both with synthetic and biological targets. In addition, the magnetic particles were, also, e$cient in removing inhibitors from clinical specimens. As this was a feasibility study, these encouraging results can be further optimized along di!erent steps of the process. Experiments with RNA targets are under way where, depending on the ampli"cation technology used, the elution step can be omitted. In conclusion, magnetic latex particles are, thus, useful as puri"cation and concentration tools in a sample preparation step before ampli"cation technologies in the diagnostic "eld.
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