High Affinity Nano Magnet for Non-Invasive Prenatal Diagnosis (Ensur)

摘要

INTRODUCTION: Prenatal screening and genetic testing are some of the most utilized and important tools in the obstetrics community. The information obtained from prenatal testing about the viability and health of an unborn child is critical to the emotional state of the parents and assists in determining clinical treatment options. The ability to accurately identify genetic abnormalities as early as possible in pregnancy allows parents and physicians to make more informed decisions and reduce the expense and emotional trauma associated with detection of genetic abnormalities later in pregnancy. Currently, only high-risk pregnancies are normally considered for amniocentesis or chorionic villus sampling (CVS). The current gold standard for prenatal diagnosis is Amniocentesis, where a small amount of amniotic fluid from the mother is removed by a syringe and is tested for genetic anomalies. The invasive nature of this procedure poses a threat to the health of the fetus as well as the mother. An ideal prenatal genetic test would be non-invasive and would have a degree of reliability equal to or greater than the invasive test. In addition, a test that could be performed much earlier in the pregnancy is extremely desirable. The test also needs to be comparable in price or less expensive than current methods. Presently, vaginal swabs from the mother are collected to extract the fetal cells using a magnetic filtration process. These methods are time consuming and laborious and suffer from low recovery. The presence of mucous in the samples also makes the isolation of trophoblast cells more difficult. To counter these issues, we have developed a High Affinity Nano magnet (HANM) sensor platform (ENSUR) for efficiently separating as well as recovering trophoblast cells from the vaginal swabs in less than 3 hours. The separation is conducted by selectively targeting trophoblast cells which express HLA-G receptors. "ENSUR" is a non-invasive assay for selective isolation of fetal cells from maternal cells utilizing a vaginal swab. It consists of a dual-core magnetic nanoparticle with high affinity toward fetal cells. Magnetic separation facilitates easy isolation of fetal cells from those of the maternal cells. The isolated fetal cells will be subsequently examined using any desired DNA or cell testing method to identify chromosomal abnormalities. METHOD: In our work, we synthesized HANM (25nm) functionalized with anti-human HLA-G antibody. Validation of our method was performed on (i) Jeg3 cells (HLA-G +ve) spiked with SkBr3 cells (HLA-G -ve), (ii) Jeg3 cells spiked in swab samples from Non pregnant females as a control (n=5), and (iii) term placenta and vaginal swab samples from pregnant women within the age of 18-37 years and gestational age of 5 to 26 weeks (n=15). In our process, nanomagnets were allowed to bind to target receptors in 1X PBS for 3 h at 37 degrees C for followed by cell separation using a magnet (pull force of 57lbs). The recovered cells were counted using an automated algorithm and stored for further genetic analysis. RESULTS and CONCLUSION: Our HANM is optimized for maximum conjugation of anti-human HLA-G antibody with negligible non- specific binding towards non-target cells (epithelial). Our results demonstrate 70% recovery of Jeg3 cells (HLA-G +ve) for every 100 spiked cells and around 50% for every 10 cells spiked with a minimum capture efficiency of 5 Jeg3 cells. Furthermore our sensor was able to efficiently capture trophoblast cells from the swab and placental samples with a significant recovery of 50% required for clinical evaluation. Our data provides a proof of concept for efficient capture of trophoblast cells and detection of Trisomy 21 in less than 24 h.