Fetal Chromosomal Abnormalities Detection Chip: A Combination of

Circulating Fetal Cell Capture and Fetal Genetic Testing

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● Clinical Background● Current Technology● Issues with Current Technology● FETAL-Chip by Zhang et al. ● Proposed Solution● Fabrication● Implementation● Testing● Risks/Limitations● Biocompatibility ● Applications● Summary

Overview

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Congenital Defects

● Result of alterations in chromosomes or genes, alters child development● Different types: single gene, chromosomal abnormality, multifactorial,

teratogenic● Early diagnosis allows for:

○ Potential interventions (ex: fetal surgery for spina bifida)○ Planning for a child with special needs

3Risk increases with maternal age (>35 y.o.)

303,000 newborns die within 4 weeks per year worldwide

Chromosomal abnormalities affect 9 in 1,000 live births

Lifelong impacts on individuals and families

Amniocentesis and Chorionic Villus Sampling (CVS)

Most Common Methods

Amniocentesis

CVS

https://www.pregnancyhealth.net/chorionic-villus-sampling-cvs-procedure-risks/4

CVS

Sample of CV is removed from placenta

Provides oxygen and nutrients, removes waste

Shares baby’s genetic makeup

9-14 weeksAmniocentesis

Sample of amniotic fluid

Protects and surrounds baby during pregnancy

Contains fetal cells and proteins

14-16 weeks

Serum Screening and NIPTs

● As early as 9-10 weeks● Uses maternal blood

sample ● Detect chromosomal

abnormalities Alternative Methods

Serum Screening

Noninvasive Prenatal Tests

(NIPTs)

https://www.bbc.com/news/health-319415385

Problems with Current Technology

● Difficult to detect and separate

● Limited accuracy● Further diagnostics

needed

Serum Screening

Noninvasive Prenatal Tests

(NIPTs)

● Invasive● High risk of

miscarriage

Amniocentesis (1 in 300 to 1 in

500)

Chorionic Villus Sampling (CVS)

(1 in 100)

Less than 6 cells per mL

with 10^9 maternal cells

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FETAL-Chip by Zhang et al.

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Frequency Enhanced Transferrin Receptor AntibodyLabelled Microfluidic Chip

● Enriches and identifies circulating fetal cells from maternal blood

● Increased capture efficiency● Decreased nonspecific adsorption as early as 7 weeks● Mitigates risks of previous techniques

Circulating Nucleated Red Blood Cells (cNRBCs)

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Well differentiated with a very limited lifespan

Vanish after parturition or pregnancy termination

Mononuclear cells with special markers

Present throughout the entire pregnancy

Complete genetic information Proteins for fetal analysis

Express special membrane-bound markers (e.g. fetal erythrocyte transferrin receptor (CD71) and glycophorin A (GPA)) which allow immune-enrichment.

Ideal targets for non-invasive prenatal diagnosis

Size-dictated Immunocapture Chip (SDI-chip)

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Capturing circulating tumor cells with antibody coating microposts (EPCAM)

Decreases non-specific adsorption of other blood cells

Original use

Isolate fetal cells with immobilized transferrin receptor antibody

Apply methodology

Proposed Solution

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Improve capture efficiency and background maternal cell reduction

2● Alter geometry:

staggered pyramids● Extra receptor:

glycophorin A (GPA)

Combine FETAL-Chip technology with diagnostic chip1

● DNA adsorption channel: isolate DNA

● PCR chip: amplify DNA● DNA microarray:

diagnose defects

Fabrication - FETAL Chip Improvements

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Front ViewPDMS Binding Sites

Fabricate Silicon MicromoldCreate Negative of desired PDMS channel using standard SU-8 silicon wafer and standard etching/resist application techniques

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Pour and Cure PDMS1. Pour liquid PDMS onto SU-8 micromold 2. De-bubble PDMS to create transparent layer3. Spin wafer to ensure even PDMS thickness4. Allow PDMS to fully cure before mold removal

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Treat Surfaces for Cell Capture1. MPTS Solution - 1 Hour + Drying

2. Crosslinker GMBS Solution - 30 Minutes at RT3. Streptavidin/Glycophorin A Solution4. Biotinylated anti-CD71 Antibody

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Bond Channel HalvesTreat bonding surfaces of the PDMS with oxygen plasma and irreversibly bond two channel halves to form an enclosed corridor for blood to flow

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Cell Capture Geometry: Offset Truncated Pyramids

Fabrication - DNA Adsorption Channel

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Etch Silicon WafersFabricate using standard etching/resist application techniques with the same geometry as the PDMS channel halves used in the FETAL chip

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Direct Bond Silicon Wafers Treat bonding surfaces of etched silicon wafers with argon plasma and attach two halves to form an enclosed silicon channel

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Preload Channel with Guanidium HCl3Fill enclosed channel with GuHCl based loading buffer to allow for DNA adsorption to silicon channel walls

In the presence of a solution with high ionic strength DNA will adsorb onto the walls of the silicon channel. The DNA can then be washed and later eluted for processing.

Front ViewSilicon Binding Sites

DNA Separation by Silicon Adsorption● Commercial DNA separation can not be

integrated into a lab-on-chip apparatus○ Hands-on, large equipment, etc;

● DNA will bind to silicon surfaces in the presence of certain salts

○ Easily miniaturized○ Easy to fabricate silicon channels

with current technology

Fabrication - PCR Chip/DNA Microarray

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Focus on single gene and multi-gene

Multi-Gene (Predictive/Comparative)Single Gene (Presence/Absence)

SRY on Y chromosome

(determines if baby is a boy)

Cystic Fibrosis, Spinal Muscular Atrophy, etc.

Cancer genes for increased susceptibility (BRCA1, BRCA2)

Like carrier screen -compare fluorescence levels (ex: polycystic

kidney disease)

Implementation - Process Flow

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DNA Adsorption Channel

FETAL Chip PCR Chip DNA Microarray

Amplified ssDNA

(Bound to fluorophores)

WasteMaternal Cells

Maternal Blood

WasteCell components

other than DNA (e.g. proteins)

Fetal Cell DNAFetal Cell

Components(Post Lysis)

Testing and Validation

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1

Find Subjects

Expectant mothers who have had

amniocentesis or CVS performed

Blood Draw

Collect maternal blood sample for enrichment and

testing

Process

Enrich and test maternal blood

sample containing fetal cells

Compare

Compare the results obtained by both

methods to determine accuracy

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Physical Risks vs. Ethical Risks

RisksPossibility of:● False positives/negatives● Damage to fetal

DNA/chromosomes

Limitations

● Chromosomal abnormalities may be difficult to detect

● Amount of protein present may still be too small to detect

● Denaturing of proteins

No major physical risks to mother or child (blood draw)

Risk of obtaining too much information about genetic composition of the child

Biocompatibility

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Only contact to human body is maternal blood draw

No risk to individuals being tested besides momentary discomfort from blood draw

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Maternal blood does not coagulate when in

contact with PDMS

Allow blood to flow through the chip for

detection

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Device components (i.e. lysis buffer) are only used in the chip

Lab on a chip device, only extracted fetal cells are in contact with device (i.e. only cells within the chip are lysed)

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Applications

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Our Device:● Noninvasively diagnose

before birth ● Identify disease causing

upregulation

Future:● Fetal proteins

(misfolding/abnormalities)● Less damaging methods

(chromosomal abnormalities)

Summary/Conclusion● Combination of FETAL-Chip and

diagnostic chip with improved capture efficiency

● Noninvasive diagnosis and advanced screening

● Wide range of data can be compiled from device

● Future applications to protein and chromosomal abnormalities

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Questions?

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ReferencesAbnormal Development - Genetic Carrier Testing. (n.d.). Retrieved from

https://embryology.med.unsw.edu.au/embryology/index.php/Abnormal_Development_-_Genetic#Carrier_TestingAmniocentesis. (2016, September 02). Retrieved from https://americanpregnancy.org/prenatal-testing/amniocentesis/Amniocentesis. (2019, March 08). Retrieved from https://www.mayoclinic.org/tests-procedures/amniocentesis/about/pac-20392914Choice in genetic testing in your first and second trimesters. (2017). Retrieved from Integrated Genetics - LabCorp Specialty Testing Group:

https://www.integratedgenetics.com/patients/pregnancyChorionic villus sampling. (2019, March 08). Retrieved from https://www.mayoclinic.org/tests-procedures/chorionic-villus-sampling/about/pac-20393533DNA Microarrays. (n.d.). Retrieved from https://www.nature.com/scitable/definition/microarray-202Dyes, probes and chemistries in real-time PCR detection. (n.d.). Retrieved from http://dyes.gene-quantification.info/Field, R. M., Kymissis, I., Shepard, K. L., & Haig Norian. (2014, August 15). An integrated CMOS quantitative-polymerase-chain-reaction

lab-on-chip for point-of-care diagnostics. Retrieved from https://pubs.rsc.org/en/Content/ArticleLanding/2014/LC/C4LC00443D#!divAbstractGenetic disorders of the fetus. (n.d.). Retrieved from https://women.texaschildrens.org/program/high-risk-pregnancy-care/conditions/genetic-disordersInheritest® carrier screen. (n.d.). Retrieved from https://www.integratedgenetics.com/inheritest-carrier-screen-comprehensiveOhnesorg, T., Vilain, E., & Sinclair, A. H. (2014, January 31). The Genetics of Disorders of Sex Development in Humans. Retrieved from

https://www.karger.com/Article/Fulltext/357956Single-gene screening. (n.d.). Retrieved from https://www.integratedgenetics.com/patients/pre-pregnancy/all-single-geneSRY gene deletion. (n.d.). Retrieved from

https://www.oumedicine.com/department-of-pediatrics/department-sections/genetics/clinical-genetic-laboratories/clinical-laboratory-services/chromosome-analysis/fish-analysis/sry-gene-deletion

Tan, Chee, S., Yiap, & Chin, B. (2009, November 30). DNA, RNA, and Protein Extraction: The Past and The Present. Retrieved from https://www.hindawi.com/journals/bmri/2009/574398/

The surface area and the volume of pyramids, prisms, cylinders and cones (Geometry, Area) – Mathplanet. (n.d.). Retrieved from https://www.mathplanet.com/education/geometry/area/the-surface-area-and-the-volume-of-pyramids-prisms-cylinders-and-cones

VistaSeq Hereditary Cancer Panel. (n.d.). Retrieved from https://www.integratedgenetics.com/test-menu/39526/vistaseq-hereditary-cancer-panelZhang, H., Yang, Y., Li, X., Shi, Y., Hu, B., An, Y., . . . Yang, C. J. (2018).

Frequency-enhanced transferrin receptor antibody-labelled microfluidic chip (FETAL-Chip) enables efficient enrichment of circulating nucleated red blood cells for non-invasive prenatal diagnosis. The Royal Society of Chemistry, 2749-2756

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