FAQs- Technology
What is Biocept’s unique technology (CEE™)?
The CEE™ technology utilizes a microfluidic device for capture, enrichment and isolation of rare cells from biological fluids. The CEE™ platform is a microfluidic channel with about 9,000 transverse, randomly sized, randomly positioned posts. As samples are processed through channels, rare cells of interest are captured on the interior surface, mostly on the posts. Post size, shape, and relative position distribution have been optimized such that the randomization of these elements prevents straight-line, regularized streamline flow, assuring maximum collisions between posts and cells.
CEE channels are manufactured out of PDMS, using standard soft lithography MEMs manufacturing techniques and are bonded to a glass coverslip. The inner surface of the microfluidic channel is coated with streptavidin tethered to the PDMS via long PEG chain. All channels are exact replicates, which becomes useful during post capture analysis where captured cell positions are recorded.
Biocept uses specially designed microfluidic pumps to improve CEE channels’ performance. A customized syringe pump, accepting up to 10 channels, was custom designed at Biocept. CEE Channels are attached to channel holding racks (also designed by Biocept) which maintain channel at an angle optimized for capture, and each CEE channel is connected through the outlet to the syringe pump via Teflon tubing. All 10 syringes are arranged radially around a central barrel, which is locked, calibrated, and pulls equally on all syringes simultaneously. This minimizes variability in flow between channels and creates a smoother flow.
How is Biocept’s patented technology different from others?
In order to maximize interactions between posts (capture elements) and cells, flow through the channel has to be disrupted, which prevents regularized flow. Many microfluidic devices use regular arrangement and spacing of the posts, which does not cause disrupted flow. Under these conditions, many more posts and closer packing of posts are needed in order to maximize cell capture. Biocept achieves disrupted flow by using a controlled random arrangement of posts size, distribution and spacing between posts.
Instead of pre-coating channel surface with a selection of antibodies, Biocept channels are coated with streptavidin. Before encountering the channel, samples are incubated with a selection of biotin conjugated antibodies. This offers an important element of flexibility, as customized antibody cocktails could be optimized for specific applications. Once inside the channel, rare cells, now tagged with biotin conjugated antibodies, are captured on streptavidin coated surfaces based on biotin-streptavidin interaction. The biotin-streptavidin bond is faster and stronger than an antigen-antibody bond, which further improves capture.
Plenty of space is left available between the posts in order to minimize channel blockage. Cell slumps and debris can create blockages in channels, especially if posts distribution is dense. These blockages interfere with flow, collect nonspecific white blood cells, and can interfere with staining. Biocept's technology avoids these issues.
How does the microfluidic channel work? How does it provide an improved captured yield?
CEE channels utilize disrupted flow, concept of rolling cell recruitment, and streptavidin-biotin interaction to capture rare cells. As cells flow through channels at high flow rates, they are forced onto the surface of posts. Both the disrupted flow and the high flow rates are needed to maximize the chance of cells encountering posts.
Once on the surface of the post, cells slow down, and roll along the surface in the direction of overall flow. While rolling, cell membranes come into contact with surfaces of posts.
Rare cells, which have previously been incubated with antibody cocktails, adhere to posts through biotin-streptavidin interactions. The rest of the cells roll off the posts without adhering and exit the channel without being captured. Those non-specific cells that remain are later washed out during a post capture processing.
Explain the three-dimension channel benefits versus a two-dimension slide
Total surface area of posts in the channel exceeds that of a microscope slide. During their passage through the channel, all cells come into direct contact with the surface of posts. On a microscope slide, given the large total number of cells, a much greater surface area is needed in order to spread out all the cells without overlapping. Furthermore, since the inner volume of the channel is only 20 microliters, relatively low volume of reagents are needed for post capture treatments.
How does CEE allow for molecular analysis? How are the FISH and PCR different?
CEE channels are compatible with most molecular essays with minimal protocol optimization. Channels are made from PDMS, which is compatible with most laboratory reagents.
For immunohistochemical analysis, captured cells undergo washing, fixation and staining. Specific steps are optimized for different applications. For FISH, steps are similar, with additional steps allotted for probe addition. Typically FISH hybridization occurs overnight, following a stringent post FISH wash. PDMS in CEE channels is thermally stable, and easily handles the range of FISH temperatures.
Samples processed for IHC and FISH are analyzed via florescent microscope. Cells are imaged directly in the CEE channels. Channels’ dimensions allow them to fit standard microscope stages as well as higher throughput automated microscope stages. Channels are imaged inverted, through the coverslip. Neither the thickness of the coverslip nor the PDMS interfere with analysis.
For PCR based applications, cells are removed from channels after capture. Removed cells are then lysed and processed according to an appropriate PCR process.
How does the CEE minimize white blood cell background?
White blood cells can nonspecifically adhere to surfaces of microfluidic devices. CEE has a threefold approach to minimize background cells. The interior of the channel itself is highly resistant to white blood cell adherence due to a layer of polyethylene glycol (PEG) grafted to the surface of PDMS. During the cell capture process, clumps of cells and debris mostly pass through the channel due to wide spacing between the posts. This further minimizes nonspecific capture. Finally, CEE-Save™ reagent is used to treat the blood, interfering with mechanisms that cause white blood cell adhesion to the device.
How does the CEE utilize an antibody cocktail and why? How were the antibodies selected?
Some microfluidic technologies use antibodies immobilized on channel’s surface. This limits the effectiveness of antibody selection, since multiple antibody combinations aren’t possible in this mode. When multiple antibodies are immobilized on channels, their effect isn’t cumulative since each antibody dilutes the other. This limits the use of antibodies to one per channel. Furthermore, different antibodies, and even different clones of the same antibody, have different kinetic properties. Some have faster reaction times than others.
Using antibody cocktail, in solution, allows Biocept to use multiple antibody combinations, specifically tailored for individual applications. Cocktail antibodies are tagged with biotin, and since incubation occurs in solution for half an hour, individual kinetic differences between antibodies are eliminated. All antibodies, once bound to cell antigens, now present the same kinetics and strength of biotin/streptavidin interaction. Unlike channel immobilized antibodies, the effect of solution based antibody cocktails is additive. Each bound antibody increases rate and strength of capture.
Antibody cocktails are selected by literature searches and empirical testing. For each application, antibodies are selected to reflect antigens expressed on cells of interest. After antibody types are selected, they are tested for cross-reactivity, specificity, stability and effect on non-specific background. All antibody cocktails undergo extensive quality control through FACs and channel capture.
What is CEE-Enhanced™ and how does it provide better staining?
CEE-Enhanced takes advantage of the biotin-conjugated antibody cocktails used in Biocept’s assays. Since only a small portion of cell’s membrane contacts the surface of the post upon capture, majority of cell membrane bound biotins are available for CEE-Enhanced. A streptavidin-dextran-fluorofor conjunct is used to label cell bound antigens and enhance fluorescent stain on candidate cells. This is especially important since not all CTCs will be cytokeratin positive (a gold standard stain in CTC field). As CTCs undergo the epithelial to mesenchymal transition, cytokeratin is often down-regulated and can not be detected using the anti-cytokeratin stain alone.