Branched DNA

Deoxyribonucleic acid (DNA) is a nucleic acid with a double-helix structure and consists of four kinds of deoxyribonucleotides. Based on long-term research, DNA synthesis has been very mature and can be operated quantitatively. Besides, characterized by nontoxic and biocompatible properties, DNA is very ideal for self-assembled process. With the myriad of tools available to manipulate DNA, there is great potential for using DNA as a generic instead of a genetic material.

Figure 1. Branched DNA.

Using different shapes of DNA as basic building blocks, self-assembled DNA can be incorporated into larger structure in a controlled manner and then is widely used in nanomaterial technology. Also, branched DNA has plenty of modifiable base sequences which link with different functional groups, such as DNA aptamers, magnetic nanoparticles and fluorescein. These advantages can applied into our design so that CTC capture can be achieved by means of specific recognition.

 

DNA Aptamer

Aptamers are single-stranded DNA, RNA, or modified nucleic acids isolated through SELEX (Systematic Evolution of Ligands by EXponential enrichment). On the basis of the unique secondary or tertiary structures formed by single-stranded oligonucleotides, aptamers can bind to a great variety of targets such as small molecules, proteins, cells, and even tissues with high affinity and selectivity.

Currently, most of EpCAM-based diagnostic and therapeutic strategies rely on anti-EpCAM antibody, in particular for CTC capture and detection. However, the use of the antibody is restricted due to its large size and instability and causes interference with target binding ability and immune response. Therefore, a smaller, cheaper, more stable and effective EpCAM binding ligand is needed for cancer diagnosis and we design the EpCAM-binding aptamer. Aptamers have longer shelflives and can be easily conjugated to nanomaterial or surfaces for subsequent cell isolation. Furthermore, aptamers also have several other attractive features: low molecule weight, easy and reproductive synthesis, lack of immunogenicity, and low toxicity. These advantages make aptamers ideal alternatives to antibodies for a variety of applications.

Figure 2. EpCAM aptamer.

 

EpCAM

Epithelial cell adhesion molecule (EpCAM) is transmembrane glycoprotein that mediates epithelial-specific intercellular cell adhesion and is involved in cell signaling, migration, proliferation, and differentiation. EpCAM is a glycosylated, 30- to 40-kDa type I membrane protein and consists of an extracellular domain, a single transmembrane domain, and a short intracellular domain. EpCAM on the surface of one cell binds to the EpCAM on a neighboring cell thereby holding the cells together. In fact, EpCAM is expressed in a variety of human epithelial tissues, carcinomas, and progenitor and stem cells.

Figure 3. Transmembrane molecule EpCAM.

EpCAM is overexpressed in most solid cancers because Wnt signal transduction pathway is activated. Therefore EpCAM represents a dominant tumor antigen candidate on the surface of circulating tumor cells (CTCs), which have been identified as the potential origin of intractable metastatic disease. In certain tumor types, overexpression has been linked to advanced stages of disease and poor overall survival, suggesting EpCAM as a potential prognostic marker and as a potential target for immunotherapeutic strategies.

In our project, we use EpCAM as the main biomarker for identifying and capturing NSCLC CTCs.

 

Magnetic Nanoparticles

Magnetic nanoparticles are uniform, mono-dispersed, spherical, core-shell superparamagnetic beads which consists of a nanometer-scale iron oxide core completely encapsulated by a high purity silica shell. Magnetic nanoparticles can be manipulated using magnetic fields. The silica shell can be easily modified with various surface functional group via covalent bonds between organo-silane molecules and silica shell.

Figure 4. Magnetic Nanoparticles.

Our NanoHunter technique is magnetic separation which use which uses magnetic beads coated with branched DNA that target an epithelial cell marker such as EpCAM to enrich CTCs.

 

Fluorescence quenching

Fluorescence quenching is a phenomenon where the intensity of fluorescence decreases due to the interaction between fluorescent molecules and other molecules. This technology is hypoallergenic and harmless to cells and plays a great role in analysis, analytical science, life science and other field as a consequence of its good selectivity, high sensitivity, reasonable prices and simple means of detection.

Figure 5. FRET.

Fluorescent dye could emit visible fluorescence. After absorbing the ultraviolet or visible light, the fluorescent dye can transform short wavelength light into the visible light wave with longer wavelength, and the light is bright. Therefore, we utilize fluorescent rhodamine, fluorescent gene FAM and quenching gene TAMRA conjugating with branched DNA. Based on the precise change of fluorescence intensity, we can identify the capture rate of circular tumor cells effectively.