In-vitro diagnostic assays are used to diagnose infection or disease by specifically targeting a unique surface antigen or DNA sequence. Traditionally it was necessary to increase microbe density by culturing the sample for a few days under strict laboratory conditions. This was an important step toward obtaining enough genetic material for accurate analysis by qPCR.
These procedures changed once magnetic nanoparticles and magnetic separation were introduced. Magnetic particles are easily modified with functional groups to bind ligands and oligonucleotides. These allow specific capture and identification of microbes. Once bound to the magnetic particles the target microbe concentration is enriched by magnetic separation. Some of the most modern techniques couple fluorescent or chemiluminescent probes to the magnetic particles to allow for real-time quantitative microbe identification or capture. These biosensors have great potential for use in clinical settings for rapid diagnosis of disease at the point of care. Microfluidic channels and lab-on-a-chip devices will continue to advance these techniques throughout the next decade. The key to making this happen is to understand what kinds of IVD coatings can be used to functionalize magnetic particles.
IVD coatings help magnetic particles bind proteins or nucleic acids
Typical examples of IVD systems involve specific identification of either surface proteins (when the microbial cell is intact) or specific DNA or mRNA sequences (when the microbial cell is lysed). In general, magnetic particles are coated with silica or a polymer matrix to prevent irreversible agglomeration. Functional groups are chemically attached to this surface to help build up the IVD coating.
Common functional groups and their use in IVD coatings:
- Epoxy groups: hydrophilic bead allows covalent binding to primary amino and sulfahydryl groups in proteins and peptides.
- Tosyl groups: hydrophobic bead allows covalent binding to primary amino and sulfahydryl groups in proteins and peptides.
- Carboxylic acids: hydrophilic bead allows covalent amide bond formation with primary amino groups in proteins and peptides through N-terminal coupling. Also binds nucleic acids
- Amine groups: hydrophilic bead allows covalent binding through reductive amination of aldehydes, which targets specific carbohydrate moieties can also be used to bind other reactive groups by coupling different crosslinking reagents. Also binds nucleic acids.
All of the above functional groups can be used to target specific antibodies, antibody fragments, proteins, carbohydrates, antigens, and enzymes. The carboxylic acids and amine groups can be used to bind nucleic acids or oligonucleotides for capture of specific genetic sequences.
An example of an IVD coating for protein detection
Staphylococcus aureus (S. aureus) poses a major threat to public health as it emerges as an antibiotic resistant bacteria that can cause deadly toxicity once it colonizes the bloodstream. A rapid method to detect S. aureus in a whole blood sample can help prevent the spread of infection. One IVD assay for S.aureus detection uses lysozyme (LysE35A) –coated magnetic particles. The magnetic particles are coated with tosyl groups, which bind the lysosyme to the bead surface. The lysozyme specifically binds S. aureus bacteria when incubated with contaminated blood samples. This system is useful at low concentrations of bacteria in whole blood because the bacteria-bead conjugates are magnetically collected to enrich the sample for qPCRwithout requiring cell culture.
An example of an IVD coating for nucleic acid detection
In an IVD assay for Enteric fever, specific primers were designed with complementary sequences to Salmonella typhi, the bacterium that causes the disease. These mRNA oligonucleotides were attached to the surface of magnetic particles. The functionalized magnetic particles were incubated with lysed whole blood samples to allow specific binding to bacteria RNA. The beads were captured by magnetic separation, which enriched the target bacteria RNA in the sample, and allowed specific detection of the bacteria by microPCR.