Non-Invasive Preimplantation Genetic Testing: Cell-Free DNA Detection in Embryo Culture Media Using a Plasmonic Biosensor.

In vitro fertilization (IVF) faces challenges in evaluating embryo quality and in determining the genetic health of embryos. Key biomarkers in the culture medium, including nucleic acids and proteins, offer promising avenues for noninvasive assessment. However, small sample volumes, low biomolecule concentrations, and potential contaminants complicate the reliable detection of genetic indicators. In this context, we developed a noninvasive preimplantation genetic testing (niPGT) approach using a superparamagnetic particles-enhanced surface plasmon resonance (SPR) imaging biosensor capable of detecting single-point mutations in nonamplified cell-free DNA released into the spent culture medium during early embryo development. Magnetic beads with a biotinylated locked nucleic acid sequence capture target sequences with single-nucleotide variations in the β-globin gene related to β-thalassemia. The assay discriminates between normal and mutated cell-free DNA evaluating their hybridization with peptide-nucleic acid probes. Our assay detects heterozygous or homozygous mutated DNA in spiked medium blank and spent culture medium at attomolar levels (1.5 pg μL-1, ∼0.75 aM) with minimal manipulation and no dilution. Only 10 μL of sample volume is needed for each analysis, providing reliable results within 2 h. This plasmonic-based test enables a rapid, biopsy-free evaluation of the embryo's genetic status, helping to identify unaffected embryos with greater confidence and supporting informed selection for implantation.