Introduction

The principle of medical diagnostics is based on the detection and quantification of chemical (cholesterol, urea) or biological molecules (antigens, viruses, bacteria, etc.) in samples of blood, urine, cerebrospinal fluid, and cellular extracts. These samples are complex media comprising high concentrations of salts and different biological molecules among which is sought the analyte. The diagnostic is carried out in two steps: (1) the capture step consists of immobilizing a biological molecule on a solid carrier capable of recognizing selectively the analyte; (2) after eliminating the residual serum, the analyte is quantified by enzymatic reaction, chemiluminescence, or radioactivity; this is called the detection step. Research and development in vitro diagnostics henceforth aim at satisfying the following four requirements: specificity, sensitivity, rapidity, and cost.

The in vitro tests currently carried out in analysis laboratories are based on the specific recognition of antigens and antibodies. These immunoassays consist of dosing either the antibodies produced by the human body in response to an antigen or the antigens themselves. The diagnosis can thus be carried out only once if a sufficient concentration of antibody has been generated by the organism. In case of infectious diseases such as AIDS (acquired immunodeficiency syndrome) or hepatitis where early detection and regular monitoring are re quired, such tests are not satisfactory (it takes the human body 3 months to generate anti-HIV antibodies associated with AIDS).

On the other hand, the detection of a pathogenic organism via the genetic material contained in its DNA (deoxyribonucleic acid) allows for a rapid and specific diagnosis as soon as illness appears. The DNA that makes up the human genome consists of two complementary strands, whose nucleic bases interact in pairs via "stacking"-type forces and hydrogen bonds to form a double-helix structure. Strand pairing or hybridization is facilitated at neutral pH and high ionic strength. Conversely, these chains can be separated and denatured by increasing temperature and pH, which break the hydrogen bonds.

Genetic diagnoses make use of DNA's property of molecular recognition. A fragment of the DNA of a given virus can be captured by hybridization with a complementary sequence immobilized onto a specific support. These specific assays became a reality principally because of milestone development of molecular biology techniques such as the polymerase chain reaction (PCR), a powerful method for amplifying small quantities of DNA extracted from the genetic material of the virus. Another key improvement was the possible synthesis of short DNA fragments (oligodesoxyribonucleotides, ODNs) using an automatic synthesizer.

A key parameter in diagnosis assays is the support used to extract the targeted biomolecule (capture step). Colloids rather than flat solid supports are generally preferred, the former exhibiting very large specific surface and being easily separated from the biological medium by centrifugation, filtration, or magnetic separation. Among these, colloid polymers attracted major interest because they can be tailored to fit the requirements of the targeted application (concentration of biomolecules, solid support in biomedical diagnostics, drug targeting, etc.) by controlling the particle size and their surface characteristics (polarity, charge, presence of reactive functions such as amine, carboxylic acid, and aldehyde groups).

The immobilization of DNA fragments or ODN on solid supports may be achieved either by physical adsorption or via chemical grafting. In the last few years, several works have focused on the adsorption of ODNs on latex particles. A priori, this method of immobilization is simpler because it only depends on the intermolecular forces existing between the solid surface and the ODN molecules. Furthermore, it is possible to control this adsorption process by varying the conditions of the medium, e.g., by adding divalent ions such as Mg2 + and Ca2 + to induce ion bridges in DNA/anionic particles systems. However, adsorption process shows two major drawbacks: (1) the immobilizing process is reversible (desorption of molecules with time, competitive adsorption with surfactant, instability of the latex/ODN conjugate); (2) ODN conformation may affect the capture efficiency of the targets via the hybridization process.

ODN grafting on the functionalized latex surface contrarily presents the benefit of creating stable latex-ODN conjugates. In addition, medium conditions can be chosen so that the ODN protrudes in aqueous phase (desorption buffer) to permit fast and quantitative hybridization/capture of DNA. Few studies of ODN grafting onto latex carriers have been reported in the literature, partly for industrial property reasons. One of the most frequently applied techniques consists of immobilizing biotinylated ODN onto latex particles bearing adsorbed streptavi-din molecules (biotin-streptavidin complex is extraordinarily strong). Chemical grafting is another way to immobilize ODN bearing a functional spacer arm (amino-link spacer) onto reactive colloidal particles. Particular mention is made of the work of Kawaguchi et al. [1], who describe chemical grafting of ODN onto poly(glycidyl methacrylate)-containing polystyrene latex particles and their utilization for specific capture of DNA fragments.

The global strategy designed for preparing colloid-nucleic acid conjugates for the purpose of diagnostic application is divided in three steps as illustrated in Fig. 1 for amino-containing latex particles and ODN-bearing amino-link spac-

FIG. 1 Methodology used for the covalent grafting of dT35 onto amine-containing polystyrene latex particles. Step 1: activation reaction of the dT35 by PDC. Step 2: adsorption and grafting of the activated dT35 onto aminated latex particles. Step 3: desorption of noncovalently grafted dT35 molecules.

FIG. 1 Methodology used for the covalent grafting of dT35 onto amine-containing polystyrene latex particles. Step 1: activation reaction of the dT35 by PDC. Step 2: adsorption and grafting of the activated dT35 onto aminated latex particles. Step 3: desorption of noncovalently grafted dT35 molecules.

ers: (1) activation step of the functionalized ODN, (2) adsorption and covalent of the activated ODN onto considered colloidal dispersion, and (3) desorption study to control the release of nonchemically grafted ODN.

The present chapter is divided into six separate parts where general discussion and appropriate examples combine:

1. Key properties of ideal colloidal polymer particles as a solid support of ODN for nucleic acid diagnostic purposes are described. The different synthesis processes and characterizations performed specifically on amino-containing polystyrene latexes are reviewed.

2. With a view to immobilizing ODN probes on latex particles via chemical grafting process, prior understanding of the interactions between these two entities is vital. The influence of various parameters on ODN adsorption onto latex particles are investigated as a systematic study.

3. In a third part, special conditions leading to complete ODN desorption are discussed. This part is the key step needed to discriminate between the ODN chemically grafted from those simply adsorbed.

4. This step reports on the covalent grafting of chemically activated oligonucleotides onto latex particles. The influence of various covalent binding conditions are presented and discussed.

5. The fifth part deals with the examination of ODN conformation immobilized onto reactive latex particles using small-angle neutron scattering (SANS).

6. This last part describes some fine applications of latex-ODN conjugates in biomedical diagnostic.

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