A Dumbbell-like Complex Formation for DNA Target Assay

A simple and highly sensitive test for the detection of nucleic acid targets is described. It is based upon complex formation between a small-diameter...
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Bioconjugate Chem. 2001, 12, 678−683

ARTICLES A Dumbbell-like Complex Formation for DNA Target Assay Agne´s Perrin, Thibault Martin, and Alain Theretz* Unite´ mixte CNRS/bioMe´rieux, 46 Alle´e d’Italie, 69007 Lyon, France. Received September 18, 2000; Revised Manuscript Received February 23, 2001

A simple and highly sensitive test for the detection of nucleic acid targets is described. It is based upon complex formation between a small-diameter magnetic particle and a larger and nonmagnetic particle through a hybridization reaction, what we have called a dumbbell-like complex. During the different steps, nonreacting nonmagnetic conjugates were eliminated by magnetic separation. At the end of the process, dumbbell complex number was estimated by counting under a microscope. Compared to the already described two-particle tests, our model was able to reach higher sensitivities, with a threshold typically in the amol/mL range (106 copies of HIV DNA/mL) without the need for complex instrumentation or genomic amplification reactions.

INTRODUCTION

Assays for the detection of nucleic acid targets useful in pathogen detection, identification, and susceptibility testing are increasingly used in diagnosis. Most of these use genomic amplification techniques such as polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), and ligase chain reaction (LCR). These methods are able to reach the highest levels of sensitivity, but are known to be very susceptible to contamination and inhibitors. For these reasons, many papers are now dealing with the development of highsensitivity methods which aim to avoid amplification steps. For example, authors have developed dedicated technologies and instrumentation based on piezoelectric (1) and SPR techniques (2, 3). Watts et al. (4) investigated the potential of a real-time optical sensor for hybridized DNA target quantification. They finally reached a sensitivity limit of 9.2 nM (12.1 ng). In (5), the authors conceived a DNA optical sensor. They used hybridization on a nucleic acid probe-magnetic bead conjugate to specifically extract DNA targets and, finally, used a chemiluminescent enzymatic detection which allowed them to reach the femtomole level. Another example of a DNA optical sensor was given by Piunno et al. (6), who described a hybridization bioassay on an optical fiber. Ethidium bromide was used for detection by fluorescence which allowed them to detect a minimum value of 86 ng of cDNA/mL. Other technologies including voltametric measurements were able to give good sensitivity. For instance, Hashimoto et al. (7) declared they have reached a detection threshold as low as 4 × 104 targets/mL, using a gold electrode modified with DNA probes. Flow cytometry was also used as a highly sensitive method to detect microspheres onto which stained DNA was captured (8), or to perform multiplexed DNA sequence analysis with fluorescent latex particles and oligonucleotides (9). Hakala et al. (10) detected single microparticles by time* To whom correspondence should be addressed. E-mail: [email protected].

resolved fluorometry after specific capture of photoluminescent europium chelate labeled oligonucleotides. The ultimate sensitivity, i.e., hybridization of two single nucleic acid probes, was reached with techniques such as fluorescence correlation spectroscopy (11) or total internal reflection fluorescence microscopy (12), but analysis parameters (extremely limited volumes, low flow rate, very long analysis time) are not acceptable for medical diagnosis. As can be seen, even when good sensitivity is obtained, all these methods require the use of dedicated or complex instrumentation. Alternately, the use of magnetic particles in immunoassays is expanding rapidly and is even becoming a generic technique known as ImmunoMagnetic Separation (IMS). Using this simple one-particle capture step, associated with sensitive detection methods such as chemiluminescence (13) or time-resolved fluorescence (14), authors significantly improved the sensitivity of conventional immunoassays. We also demonstrated the feasibility of a very sensitive Scanning Force Microscopic Immunoassay (SFMIA) associated with capture and detection of TSH antigens by the use of antibody-magnetic nanoparticle conjugates (15), which led to the detection of 1 amol of TSH/mL (i.e., 5 × 105 TSH molecules/mL). The presence of magnetic particles associated with a specific biological recognition event may also be evidenced by measuring parameters such as magnetic permeability (16) or magnetoresistance (17) with good sensitivity. Other simple tests using two particles and aiming to have an acceptable to high level of sensitivity had already been described. Lim et al. (18) recently described an assay based on an agglutination inhibition principle test in tubes, using a large magnetic particle and a smaller colored latex particle. Detection is based upon direct observation by the user, and the sensitivity limit was said to be 16 µg/mL of antibody (i.e., approximately 0.1 nmol/ mL). Another judicious two-particle assay was proposed by Mizutani et al. (19), who studied the detected virus captured on microbeads using antibody-labeled magnetic nanoparticles. The reaction was quantified by the analy-

10.1021/bc000115s CCC: $20.00 © 2001 American Chemical Society Published on Web 08/18/2001

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Table 1. Characteristics of Oligonucleotides Used for Dumbbell Formation reference

label

biotin-ODN1 5′ biotin HRP-ODN2 5′ HRP target none

hybridized base with number % GC MTa (°C) target target 72

32 29 41.7

43.7 27.6 67.6

56.8 47.9

a Melting temperatures (MT) were calculated by Eurogentec (Belgium).

NaCl. Particle conjugates blocking buffer was PBS, 40 mM ethanolamine, 0.05% Tween 20, and 0.5% bovine serum albumin. Particle conjugates storage buffer consisted of PBS, 0.05% Tween 20, 0.1% bovine serum albumin, and 0.05% sodium azide. Biotin binding buffer was 50 mM Tris, 100 mM NaCl, pH 7.5, and hybridization buffer was 10 mM Tris, pH 8.0, 1 mM EDTA, 1 M NaCl, 0.14 mg/mL salmon sperm DNA, and 0.05% Triton X-100. METHODS

Figure 1. Diagram of oligonucleotide hybridization onto target. Biotin-ODN1 (32 mers) hybridizes the DNA target (72 mers) at its 3′ end, and HRP-ODN2 (29 mers) at its 5′ end.

sis of interference fringes generated when focusing a laser beam onto concentrated nanoparticles: levels as low as 0.1 pg/mL of p24 antigen can be detected. Earlier, Kim et al. (20) described the same two-particle principle using a turbidimetric detection and obtained a sensitivity of 110 ng/mL of antibody (i.e., approximately 0.1 pmol/mL). To our knowledge, very little work has been published on the use of the two-particle assay for nucleic acid target detection. Recently, Bains (21) described a simple agglutination format between dense and light particles which led to the detection of 105 DNA targets/mL in 6 h, using a sucrose density gradient and ultracentrifugation for the separation and quantification of complexes. Elghanian (22) proposed a selective colorimetric assay, based on the agglutination of modified gold nanoparticles in the presence of a DNA target which enabled detection of 10 fmol/test (3 × 1012 molecules/mL) of an oligonucleotide. Here, we intend to describe a rapid and very simple two-particle test which has the advantage of using standard instrumentation and can detect DNA targets down to the attomole per milliliter range. MATERIALS

Chemicals and Biochemicals. 1-Ethyl-[3-(3-dimethylamino)propyl]carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (NHSS) were from Fluka AG (Germany). Ethylene glycol and monoclonal anti-horseradish peroxidase (anti-HRP) antibodies and o-phenylenediamine (OPD) enzymatic substrate were obtained from Sigma Chemical Co. (St Louis, MO). Additional chemicals were respectively salmon sperm DNA from Boehringer, Triton X-100 from Sigma, and Tween 20 from Merck. Oligonucleotides were obtained from Eurogentec (Belgium). The HRP modification was carried out at bioMe´rieux. Table 1 displays characteristics of oligonucleotides used throughout this paper, and Figure 1 visualizes which way the target is hybridized between both labeled nucleic acids. Particles. Superparamagnetic streptavidin nanoparticles (145 nm diameter) were from Immunicon Corp. (Huntingdon, PA). Carboxylate polystyrene latex particles (850 nm diameter, 10.1% solids content) were obtained from Bangs Laboratories, Inc. (Fishers, IN). Buffers. Particle coupling buffer consisted of 10 mM Na2HPO4/NaH2PO4, pH 6.8. Phosphate-buffered saline (PBS) was 50 mM Na2HPO4/NaH2PO4, pH 7.4, 0.15 M

Synthesis of Anti-HRP/Bangs Particle Conjugates. EDC (0.1 mg) and NHSS (2 mg) were diluted in 0.4 mL of particle coupling buffer in the presence of 0.05 mg of anti-HRP antibodies. Then 10 mg of Bangs carboxylated polystyrene latex particles was added while stirring and incubated for 1 h at 37 °C. The reaction was stopped by the addition of 0.5 mL of particle conjugates blocking buffer, followed by centrifugation to precipitate microspheres (10 min at 8000 rpm). After resuspension in 0.5 mL of the blocking buffer, centrifugation was repeated once, and anti-HRP latex conjugates were finally resuspended in 0.1 mL of particle conjugates storage buffer. They were stable for several months at +4 °C. 35 S Radioactive Labeling of Oligonucleotides. Biotin-ODN1 was labeled with 35S at its 3′ end using the labeling kit from Boehringer Mannheim (Terminal Transferase 220582) and (R-35S)ddATP from NEN. After incubation for 1 h at 37 °C, purification was carried out with Sephadex G25 QuickSpin columns also from Boehringer. The specific activity of the resulting 1 pmol/µL labeled oligonucleotide solution was measured on a liquid scintillation analyzer (1900TR) from Packard. Synthesis of Biotin-ODN1/Immunicon Nanoparticle Conjugates. Solutions containing different concentrations of biotin-ODN1 oligonucleotides were obtained by mixing 35S-labeled oligonucleotides with unlabeled (cold) oligonucleotides at known ratios. These solutions were then allowed to react for 1 h with 7 × 108 streptavidin-coated Immunicon particles in 1 mL of hybridization buffer. Excess oligonucleotides were eliminated by a series of rapid magnetizations and resuspensions in hybridization buffer. After resuspension in 0.2 mL of water, particle emission activity was measured. Knowledge of the specific activity, labeled/unlabeled oligonucleotide ratio, and remaining activity on the particles allows the calculation of the binding yield, i.e., the fraction of oligonucleotides retained on the particles. Within a maximal 8% error, no detectable loss of activity was found due to the presence of the particles, as the sum of activities measured in supernatant and on particles matched the total activity of the initial solution. Formation of Immunicon-Bangs Dumbbells. The principle is described in Figure 2. It is known that hybridization in solution gives better reaction rates than in the solid phase, since the solid support introduces a diffusion limitation which significantly reduces kinetics (23). Hence, targets and ODN were incubated first, before introducing particles. Biotin-ODN1 (1012 molecules), HRP-ODN2 (6 × 1011 molecules), and an increasing number of targets (0-1010 molecules) were incubated in 1 mL of hybridization buffer for 1 h at 37 °C. Then, 7 × 108 particles of Immunicon streptavidin/magnetic particle conjugates were added and allowed to react with hybrids for 1 h at 37 °C. Solutions were concentrated by magnetic attraction on rare earth magnets (NdFeB, diameter × height ) 12 ×

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Figure 2. Representative diagram of a dumbbell between one latex particle and one magnetic particle. First, biotin-ODN1, DNA target, and HRP-ODN2 are hybridized. Then, Immunicon magnetic particles are added for capture of hybrids. After magnetic separation, anti-HRP latex particles are incubated with the magnetic complex to form so-called dumbbells, i.e., a two-particle system for DNA capture and detection.

6 mm) for 20 min. Pellets were then resuspended with 6 mL of hybridization buffer. A total of 107 anti-HRP/Bangs particle conjugates diluted in hybridization buffer were added for a final volume of 10 µL. Incubation was carried out for 1 h at 37 °C. Dumbbells and free magnetic nanoparticles were separated from unbound latex particles by magnetic attraction for 30 min. Pellets were resuspended once with 20 µL of hybridization buffer and again isolated under a magnetic field. These were finally dispersed in 5 µL of ethylene glycol. The role of this viscous solvent was to limit the particle’s brownian motion, thereby making counting under a microscope easier. The 5 µL particle drops (V) were then deposited onto a Malassez cell defining small calibrated volumes (v) of a few picoliters. Such a cell is normally used for counting a number of cells or bacteria and turning it into a volumic concentration. After deposition of coverglasses onto the cell, latex particles were counted with an optical microscope (Olympus) equipped with a ×40 lens, inside 10 compartments giving an average number of particles per compartment (n). The total number of particles (N) in the sample was extrapolated from this value (N ) nV/v). Dumbbell Analysis by Transmission Electron Microscopy (TEM). Purified dumbbells obtained with various target concentrations were deposited onto formvar carbon film TEM grids (Ted Pella Inc.) and analyzed on a Philips CM120 microscope under a 80 kV voltage. Controls were carried out to ensure that the drying process did not induce magnetic nanoparticle adsorption onto latexes. Also, samples were analyzed before and after magnetic separation to make sure that this step does not produce false dumbbells. RESULTS

Characterization of Anti-HRP/Bangs Particle Conjugates by Size Measurement. To check that antibody grafting onto latexes did not induce aggregation, the size of grafted particles was measured by quasi-elastic light scattering using a Zetasizer 3000 (Malvern). No consequent diameter modification was measured before (883 ( 9 nm) and after coupling (905 ( 3 nm). Also, microscopic observation of conjugates revealed the absence of aggregates. Immobilization Yield of Biotin-ODN1 onto Immunicon Nanoparticles. To avoid competition between free biotin-ODN1 in excess and those hybridized with

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Figure 3. Binding yield of biotin-ODN1 onto streptavidin Immunicon particles as a function of the initial number of biotinODN1 molecules in solution estimated using radiolabeled oligonucleotides. Increasing concentrations of 35S-labeled nucleotides are incubated in the presence of magnetic particles. After magnetic separation, the radioactivity is measured directly on the resuspended particle solution, and the number of nucleotides per particle is calculated.

targets, the binding capacity of streptavidin/Immunicon nanoparticles has to be greater than the total number of biotin-ODN1 molecules. In that perspective, the use of solutions containing radiolabeled biotin-ODN1 enabled an estimation of the fraction of ODNs that remained immobilized onto the particles. This binding yield is shown in Figure 3 as a function of the initial number of biotin-ODN molecules in solution. With an initial number of 1012 biotin-ODN1 molecules, this figure indicates that the bonding yield is higher than 97%. Hence, there is no competition between free and hybridized biotin-ODN1 molecules in the dumbbell formation conditions: both are equally immobilized onto streptavidin/Immunicon nanoparticles. This biotin-ODN1/ Immunicon particle ratio will thus be used throughout the following experiments. Dumbbell Isolation Efficiency by Magnetic Separation. First, the efficiencies of magnetic nanoparticle conjugate separation and latex particle conjugate elimination were estimated separately. Magnetic Nanoparticle Recovery Yield during the Magnetic Separation Step. A total of 100% of the Immunicon nanoparticles was extracted within 10 min when diluted in 1 mL of buffer, by placing the tube against a rare earth permanent magnet. This was determined by measuring the optical density of the particle solution before and after separation at 460 nm. Latex Particle Elimination Yield. Solutions containing only latex particles at the same concentration as those used for dumbbell formation (107 particles in 10 µL) were placed in the tube. Supernatant was discarded twice, and the remaining particlesslater called washing noiseswere counted under the microscope. An elimination yield of more than 99% was obtained. Considering the initial number of particles (107), the sensitivity of the assay is thus limited to less than 105 targets by this washing noise. Separation of Dumbbells. The efficiency of dumbbell retention, in the presence of two types of particles at the same time, was estimated. The separation duration was extended to 30 min (instead of 10 min for isolated Immunicon particles) because of drag forces exerted on latex particles which are supposed to slow their motion. To evaluate a possible loss of dumbbells between two separation cycles, the number of latex particles was

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Figure 4. TEM analysis of dumbbells for increasing target numbers. (A) False dumbbells due to the nonspecific adsorption or the drying of a magnetic particle on latex (no magnetic separation or any other process). (B) No target in solution. (C) 107 targets. (D) 109 targets. (E) 1010 targets before magnetic separation. (F) Same sample as (E) (1010 targets) after magnetic separation.

evaluated in the same sample after the first and second separation steps. It was shown that as much as 50% of dumbbells can be lost in this way in the presence of 108 targets. Two possibilities can explain this loss: First, due to their low magnetic charge, some dumbbells are discarded while sucking up the supernatant, as the magnet can only exert weak magnetic forces. Second, the liquid meniscus also exerts strong capillary forces on the particle’s spot and probably carries along the same poorly magnetic complexes. Dumbbell Study by TEM. Electron microscopy is one of the few methods enabling observation of the structure of dumbbells. Controls ensure that the sample preparation drying step did not induce the formation of a large number of false dumbbells, i.e., nonspecific adsorption of one or several magnetic particles onto one latex. Contrary to the washing noise, these latex particles cannot be eliminated during magnetic washes since they are sensitive to the magnetic field as well as specific dumbbells. For this purpose, latexes, magnetic particles, and oligonucleotides were mixed briefly in the absence of targets, and immediately deposited onto grids. A few

isolated latexes were found to bear magnetic particles as shown in Figure 4A. In this figure, isolated Immunicon particles as well as latex are clearly defined. The structure of the magnetic particles is irregular and varies in shape and size. Some of them are seen on the bottom of the grid, and one seems to be immobilized on the latex. A kind of gel structure that may contain Immunicon particles is also often observed close to the latex. In any case, it was not possible to determine whether this false dumbbell was formed during the short contact time between particles before grid preparation, or during the drying itself. This phenomenon was limited, and the analytical method was not questioned. Another control was performed to ensure that magnetic attraction did not induce the formation of false dumbbells by forcing the contact between both types of particles. A sample in the presence of 1010 targets was analyzed before (E) and after (F) separation. In both cases, latex aggregates are observed, proving that magnetic attraction is not in question. In panel E, salt deposits appear since the saline buffer was not eliminated from the sample as is the case for panel F.

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Figure 5. Variation in the number of latex particles as a function of the number of targets in solution. First, biotin-ODN1, increasing concentrations of DNA targets, and HRP-ODN2 are hybridized. Then, Immunicon magnetic particles are added for capture of hybrids. After magnetic separation, anti-HRP latex particles are incubated to form dumbbells. These dumbbells and free magnetic particles are washed twice to eliminate unbound latexes, resuspended in ethylene glycol, and deposited in a Malassez cell for counting under a microscope, and finally particle numbers are determined as a function of initial DNA concentration.

Aggregate size varied as a function of target concentration. Images B (0 target), C (107 targets), D (109 targets), and F (1010 targets) demonstrate the evolution of the dumbbell format into a typical agglutination reaction. As a matter of fact, beyond 107 targets, the target/latex particle ratio is greater than 1, which led to the possibility for particles to bind to each other via several targets and several Immunicon particles. Cluster size distribution is, of course, observed in each sample. As can be seen, latex particles are linked through the intermediate of magnetic nanoparticles. This confirms the specific aspect of the reaction and that these are not agregates due to simple nonspecific adsorption between latex particles. These clusters are distinguishable from the few observed on the drying control sample. Also, a decrease in the number of isolated Immunicon particles on the bottom of the grid is observed as the target concentration increases, since more of them are involved specifically in dumbbells and immobilized onto latexes. Assay Sensitivity. Increasing concentrations of targets were assayed, and the resulting dumbells were counted under an optical microscope after depositing the sample in a Malassez cell. As a matter of fact, only the latex particles are seen and counted under the optical microscope. Magnetic nanoparticles are not visible and thus do not interfere with the measurement. Results are described in Figure 5. A weak error in particle counting can be attributed to the confusion between roughly spherical dusts and true colloids. Furthermore, the standard deviation on each concentration pointsan average of 10 measurements on the same samplesis important (20-30%) since it was not possible for the experimentor to evaluate a very large amount of particles that would statistically better represent the sample. The sensitivity threshold is defined as the minimal target concentration enabling a detection signal higher than that obtained for the background plus 3 times the standard deviation on the noise value to be reached. From this calculation, below 106 targets/mL, it was not possible to discriminate background from specific signals. About 7 × 104 latex particles were counted in this sample,

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Figure 6. Evaluation of dumbbell formation yield, i.e., the percentage of latex counted at the end of the process over the number of targets. For each target concentration, the particle number obtained from Figure 5 after background subtraction is divided by the number of targets initially introduced in the solution.

representing 0.7% of the initial quantity. This background is essentially due to washing noise (latex particles not eliminated during the washing steps) for 0.1% (estimated as described in the section Dumbbell Isolation Efficiency by Magnetic Separation, by processing latex particles alone) and to false dumbbells (nonspecific adsorption of magnetic nanoparticles onto latex microspheres) for about 0.6%. Nonspecific interactions might be due to electrostatic or hydrophobic interactions between latexes and magnetic particles. TEM was used to ensure that the magnetic separation did not enhance false dumbell formation to a significant extent. A significant increase in the number of particles is observed under an optical microscope beyond 106 targets/ mL, which represents a sensitivity threshold of 2 amol/ mL. It is also observed that the presence of latexes becomes detectable by the naked eye beyond 108 targets/ mL. The reaction yield, i.e., the ratio between the number of specific dumbbells and the number of targets in solution, was estimated from Figure 5 by subtracting the background from the number of latex particles for each target concentration, and is depicted in Figure 6. Best yields were obtained for low target concentrations, and did not exceed 5%. Such a result can be explained by method limitations described above. First of all, the capture of hybrids onto latex particles may be limited intrinsically by the affinity of the HRP/anti-HRP antibody system. Furthermore, TEM pictures showed that Immunicon particles are rough and not spherical, which probably enhances steric hindrance during the binding of the two particles. Also, the nonspecific latexessfalse dumbbells plus washing noiseslimit the sensitivity threshold. Beyond 107 targets, as seen in the TEM section, there is statistically more than 1 target per latex, and the drawing in Figure 2 showing 1 latex and 1 magnetic particle per target is not representative. Clusters of particles are formed, leading to a drop in reaction yield. DISCUSSION

As demonstrated, this assay reached a very high sensitivity threshold, in the attomole per milliliter range. It is easy to use, does not need complex instrumentation, and avoids tedious amplification techniques.

Dumbbell-like Complex Formation

However, we identified several drawbacks which currently limit the overall sensitivity. Possible approaches to further enhance the assay performance are discussed below. (a) Capture of hybrids on latex particles may be limited by the affinity of the HRP/anti-HRP antibody system. It would be worthwhile testing other systems that may have a better affinity: other antibody/antigen pairs or DNAlabeled particles. Furthermore, on TEM pictures, Immunicon particles showed a bumpy structure which may induce steric hindrance and is probably not the most suitable for dumbbell formation. We would probably benefit from working with spherical and smooth magnetic particles. Currently, other commercial particles as well those synthesized in our lab are being tested. (b) A large proportion of dumbbellssup to 50%sare lost during magnetic separation. The process has to be improved by developing specifically suited systems. Furthermore, particles with higher iron oxide contents would enable more magnetic dumbbells to be obtained, which are better retained on magnets. Nevertheless, attempts with larger and easily isolated particles such as Dynal (2.8 µm diameter) or Seradyn (1 µm diameter) gave no satisfactory sensitivity (detection threshold beyond 1010 targets/mL, data not shown). It is believed that the lower diffusion coefficient of larger linked particles limits their motion and, therefore, their binding rate onto latexes. On the other hand, testing with smaller and weakly magnetic particles from Miltenyi (50 nm diameter), used with High Gradient Magnetic Separation systems (24), led to intermediate results (107 targets/mL). In conclusion, particles easier to isolate than Immunicon, but whose size does not exceed 200 nm, should be a good choice. (c) About 0.8% of the latexes initially introduced, resulting from the nonspecific interaction between magnetic nanoparticles and latex particles, are found in the samples in the absence of targets and, thus, restrict sensitivity. The formation of false dumbbells may be limited by working on treatments to reduce nonspecific adsorption of magnetic particles onto latexes. Another possibility may be to use smaller latexes which are easier to carry in the magnetic fields and to retain on magnets while discarding supernatant. For the moment, assays in this direction show that it is necessary in this case to increase the number of latexes to maintain the same reactive surface, which finally induces an important level of background, which is harmful for sensitivity. (d) The manual detection counting was shown to induce large standard deviations in the final results. It would be profitable and less tedious for the experimentor to set up an automated system enabling particles to be counted to a larger extent. Furthermore, the use of fluorescent particles would avoid the confusion between dusts and latexes. Besides, an interesting option would be to count particles using systems based on laser light scattering. CONCLUSION

The assay described in this paper enabled a very high level of sensivity for DNA target detection (106 molecules/ mL) to be reached without the use of sophisticated technologies. With the reaction being detectable by the

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naked eye beyond 108 targets/mL, applications may be considered for the rapid and sensitive assay of pathogenic material in difficult conditions, i.e., outside the analysis laboratory. Optimizations are foreseen with the aim of pushing the sensitivity threshold one step beyond. This could allow the simple detection of pathogen nucleic acids, without the use of amplification techniques and their associated drawbacks. Applied to the identification of infectious diseases, this test could also have a high potential in early diagnosis and susceptibility testing. These results and experiments have been included in the bioMerieux patent 99/10675. LITERATURE CITED (1) Fawcett, N. C., Evans, J. A., Chien, L. C., and Flowers, N. (1988) Anal. Lett. 21, 1099-1114. (2) O’Meara, D., Nilsson, P., Nygren, P.-A., Uhlen, M., and Lundeberg, J. (1998) Anal. Biochem. 255, 195-203. (3) Pollard-Knight, D., Hawkins, E., Yeung, D., Pashby, D. P., Simpson, M., McDougall, A., Buckle, P., and Charles, S. A. (1990) Ann. Biol. Clin. 48, 642-646. (4) Watts, H. J., Yeung, D., and Parkes, H. (1995) Anal. Chem. 67, 1283-1289. (5) Chen, X., Zhang, X. E., Chai, Y. Q., Hu, P., Zhang, Z. P., Zhang, X. M., and Cass, A. E. G. (1998) Biosens. Bioelectron. 13, 451-458. (6) Piunno, P. A. E., Krull, U. J., Hudson, R. H. E., Damha, M. J., and Cohen, H. (1995) Anal. Chem. 67, 2635-2643. (7) Hashimoto, K., Ito, K., and Ishimori, Y. (1994) Anal. Chem. 66, 3830-3833. (8) Samoylova, T. I., and Smith, B. F. (1999) BioTechniques 27, 356-361. (9) Fulton, R. J., McDade, R. L., Smith, P. L., Kienker, L. J., and Kettman, J. R. (1997) Clin. Chem. 43.9, 1749-1756. (10) Hakala, H., Maki, E., and Lonnberg, H. (1998) Bioconjugate Chem. 9 (3), 316-321. (11) Kinjo, M., and Rigler, R. (1995) Nucleic Acids Res. 23 (10), 1795-1799. (12) Harada, Y., Funatsu, T., Murakami, K., Nonoyama, Y., Ishihama, A., and Yanagida, T. (1999) Biophys. J. 76, 709715. (13) Yu, H., Ahmed, H., and Vasta, G. R. (1998) Anal. Biochem. 261, 1-7. (14) Yan, K. T., Ellis, A., Montgomery, J. M., Taylor, M. J., Mackie, D. P., and Doell, S. W. J. (1998) Res. Vet. Sci. 64, 119-124. (15) Perrin, A., Theretz, A., Lanet, V., Vialle, S., and Mandrand, B. (1999) J. Immunol. Methods 224, 77-87. (16) Kriz, K., Gehrke, J., and Kriz, D. (1998) Biosens. Bioelectron. 13, 817-823. (17) Baselt, D. R., Lee, G. U., Natesan, M., Metzger, S. W., Sheehan, P. E., and Colton, R. J. (1998) Biosens. Bioelectron. 13, 731-739. (18) Lim, P. L., Tam, F. C. H., Cheong, Y. M., and Jegathesan, M. (1998) J. Clin. Microbiol., 2271-2278. (19) Mizutani, H., Suzuki, M., Fujiwara, K., Shibata, S., Arishima, K., Hoshino, M., Ushijima, H., Honma, J. H., and Kitamura, T. (1991) Microbiol. Immunol. 35/9, 717-727. (20) Lim, P. L., Ko, K. H., and Choy, W. F. (1989) J. Immunol. Methods 117, 267-273. (21) Bains, W. (1998) Anal. Biochem. 260, 252-255. (22) Elghanian, R., Storhoff, J. J., Mucic, R. C., Letsinger, R. L., and Mirkin, C. A. (1997) Science 277, 1078-1081. (23) Soderlund, H. (1990) Ann. Biol. Clin. 48 (7), 489-491. (24) Miltenyi, S., Miller, W., Weichel, W., and Radbruch, A. (1990) Cytometry 11, 231-238.

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