Related Field of The I nvention

The present invention relates to microfluidic device for selective capture of biological entities suspended in a medium and to medical diagnostics.

Background of The I nvention (Prior Art)

Cancer is the second common cause of deaths worldwide (8.8 million deaths in 2015) associated with an important economic burden (up to 4% of global GDP) .

The initial diagnosis of cancer requires invasive tissue biopsy of the tumor, an expensive (€6,000 per patient per procedure) , lengthy and painful process that carries significant risk of infection. I ts greatest limitation is that sampling of a single tumor may not capture all the mutations present, since cancer evolves genetically over time, necessitating continuous monitoring for personalized therapy. A promising solution is the liquid biopsy, which involves sampling from body fluids, mainly blood, to analyze cancer biomarkers.

Circulating Tumor Cell (CTC) enrichment/isolation systems are one of the main pillars in liquid biopsy market and expected to reach $8.7B in 2020, with CAGR of 15% (Grand View Research, 2016) .

CTCs are the cells that disseminate into blood from primary or metastatic tumors and play a crucial role in metastatic cascade. Prognostic value of CTCs has been proven and approved by FDA for breast, prostate and colorectal cancer, where the higher number CTCs (> 5 CTCs/7.5 ml blood) is correlated with lower overall survival rate (OSR) as a result of CellSearch® study.

Other potential clinical utilities of CTCs include disease monitoring, therapy guidance, patient stratification for precision medicine and personalized therapy, screening for early diagnostics, cancer research, and drug development. The main challenge in the use of CTCs in routine clinical practice arises from the difficulty of their isolation from blood as they are extremely rare (as low as one CTC in a billion blood cells) .

None of the current CTC isolation technologies can provide necessary sensitivity, reliability, robustness, ease of use and cost efficiency, which are the most important user needs in terms of clinical and economic perspectives.

There are currently around 40 companies in the competitive landscape for the CTC market. Among these, there is only one FDA-approved CTC detection system in the market, from the Janssen Diagnostic company (CellSearch® ) , recently acquired by Menarini Silicon Biosystems. The system is widely considered as the gold standard for the enumeration of CTCs of breast, prostate, and colorectal cancers. A single test costs around $450 to $600, in US and Europe. Although approved by FDA, CellSearch® is not in routine clinical use mostly due to its high cost of infrastructure and centralization in certain clinics causing sample transfer problems.

Besides, the technologies developed afterwards have reported much higher CTC numbers for the same clinical samples, decreasing the reliability of the system.

The CTC isolation platforms that utilize microfluidic technologies for immunoaffinity-based CTC isolation are IsoFlux (Fluxion Biosciences) , LiquidBiopsy (Cynvenio) , Biocept and Biofluidica.

The IsoFlux (Harb W. , et al. , 2013) and LiquidBiopsy (Winer-Jones J.P. et al. , 2014) platforms utilize Ficoll-density centrifugation for pre-enrichment and off-chip immunomagnetic labelling of CTCs in the sample before loading the sample into microfluidic chip for magnetic separation under microfluidic flow. The main drawbacks of these technologies are the long pre-processing time for sample preparation to enrich CTCs before sample loading. The latter two systems, on the other hand, utilize antibody-coated microfluidic channels for isolation of CTCs from biological fluids, similar to the method and device proposed within the frame of this invention.

The technology presented in US 9250242B2 is based on the antibody coated, parallel, narrow (25 pm) and deep sinusoidal microfluidic channels, which favors the rolling motion of the cells on the surfaces. I n contrast, the proposed invention suggests a wider channel including pillars to flow path of the biological entities.

The channel design presented in US 2006/0160243A1 is based on the antibody coated cylindrical pillars arranged randomly in a microfluidic channel . The cylindrical pillars have differing diameters. The device enhances the flow path of the biological entities so that straight- line flow is interrupted by a pattern of transverse posts. I n a similar manner, US 2014/0154703A1 encompasses methods and microfluidic device for diagnosis of cancer comprising an input, an output and an array of obstacles disposed there-between and further comprising support pillars coated with antibody. Diameter of each of the support pillars and distance between pillars can change according to the different channel regions. US 2007/0026417A1 discloses a method for detecting , enriching , and analyzing circulating tumor cells and other particles. The shape of obstacles is cylindrical. Due to the antibody-antigen interaction on pillar, cells could be captured. I n contrast to the cylindrical obstacles proposed in the mentioned previous inventions, the proposed invention herein provides a chaotic traj ectory for the biological entities by drop-shaped pillars arranged regularly inside a microfluidic channel formed by ellipse segments. All the pillars have the same dimensions and the invention increases collisions the between biological entities and the pillars by increasing the surface area and by scanning all the attack angles.

Brief Description of The I nvention

I n the present invention a m icrofluidic device, which provides selective capture of biological entities suspended in a medium is proposed.

The device enables a continuous change of the attack angle, thus keeping the chaotic trajectories of the biological entities throughout the channel.

The device includes pillars in droplet shape, resulting in increased entity/surface interaction probability to capture the target biological entities among various other entities in a suspension , which results in increase in the capture efficiency (sensitivity) . The increase in entity/surface interaction provides increase in inter-pillar distances at least three to ten times of the target entity’s characteristic dimension instead of the typical value of two to three times of the target entity’s characteristic dimension, without compromising the capture efficiency. Wider pillar distance decreases the probability of channel clogging while the sample is passing through the channel. This is especially important when working with the high concentration suspensions.

I n a particular embodiment for capturing CTCs from bodily fluids, such as blood, the increase in entity/surface interaction provides the use of 75-150 pm inter-pillar distances instead of 50-70 pm, without compromising the cell capture efficiency.

The pillars are in the same shape and distribution pattern throughout the channel that significantly reduce the design input parameters, resulting in a simplified design procedure.

Furthermore, wider inter-pillar distances provide versatility in the manufacturing processes including various polymer molding options.

The application of the invention will be on biomedical microsystems for in vitro diagnostic (I VD) or research use only (RUO) purposes. One application example is detection of biological entities, such as CTCs from the blood samples of cancer patients. CTCs in the blood sample can be selectively captured among other peripheral blood cells thanks to their distinct surface proteins, which selectively interacts with the coated antibody on the channel surface.

The device maintains chaotic trajectories for the biological entities throughout the channel without changing the pillar shape and arrangement. This is realized by utilizing a channel formed as the combination of flipping ellipse segments (successive chords facing opposite directions) connected to each other, such that the starting edge of the successive ellipse segment is located at the halfway of the chord of the preceding ellipse segment.

The inventive step is to define fluid streamlines over a uniformly distributed pillar pattern by incorporating a channel structure as described above, leading to continuously changing attack angle throughout the channel. This brings about the following advantages: • Higher sensitivity: High efficiency capture of biological entities due to higher frequency of entity/surface interaction.

• Clogging-free channel operation : The increase in entity/surface interaction provides increase in inter-pillar distances at least three to ten times of the target entity’s characteristic dimension instead of the typical value of two to three times of the target entity’s characteristic dimension, without compromising the capture efficiency. Wider pillar distance decreases the probability of channel clogging while the sample is passing through the channel. This is especially important when working with the high concentration suspensions.

• Ease of design : Simplified design procedure with significantly reduced design input parameters due to the uniformly distributed pillar pattern.

• Versatile and cost-effective device manufacturing: Wider inter-pillar distances provide versatility in the manufacturing processes including various polymer molding options.

Definition of The Figures

FI G. 1 . Depicted is schematic of the microfluidic channel segment with an array of pillars in an exemplary arrangement.

FI G. 2. Depicted is the schematic of a single pillar having a leading edge, a cambered edge, an upper surface, a lower surface and an attack angle given in exemplary ratios.

FI G. 3. Depicted is the schematic of interaction between biological entity and antibodies coated on a single pillar along the flow streamlines.

FI G. 4. (A, B, C, D) Depicted is the microfluidic device with different capture regions showing the spatial localization of biological entities captured by specific antibodies in a flat microfluidic channel. FI G. 5. Depicted is the schematic of an ellipse segment and a microfluidic channel formed by ellipse segments comprising arrays of pillars in an exemplary arrangement.

FI G. 6. Depicted is computer simulation of various flow paths of a medium through ellipse segments with an arrangement of pillars inside two ellipse segments.

FI G. 7. Depicted is photography of exemplary microfluidic channel formed of eight ellipse segments and one flat channel.

Description of The Components and Parts of The I nvention

The components shown in the figures prepared for a better explanation of the microfluidic biological entity separation enhancement device is numbered separately and explanation of each number is given below.

1 . Flow direction

2. Fluidic inlet

3. Fluidic outlet

4. Microfluidic channel

5. Pillar

6. Biological entity

7. Leading edge

8. Cambered edge

9. Upper surface

10. Lower surface

1 1 . Ellipse segments

12. Attack angle

13. Antibody coating

14. Streamlines

15. Rolling motion Detailed Description of The I nvention

The device comprises flow (1 ) , a fluidic inlet (2) and fluidic outlet (3) , and a main microfluidic channel (capture region) (4) including pillars (5) acting as obstacles in the channel (Figure 1 ) . Each pillar (5) has a droplet shape such that each one comprises a sharp leading edge (7) , a cambered edge (8) , an upper surface (9) and a lower surface (10) , wherein the leading edge (7) and the cambered edge (8) are different (Figure 2) and wherein an attack angle (12) is defined.

The inner surface of the main channel including the surface of the pillars (5) is coated with an antibody (13) suitable for the specific capture of the target entities (6) according to their distinctive surface proteins among various other entities in a suspension (immunoaffinity-based capture) .

Pillars (5) are typically used in such devices in order to increase the surface area of the microfluidic channel (4) and the collision probability of the entities (6) to the walls, both increasing the entity/surface interaction, thus the capture efficiency of the device (Figure 3) .

However, if the microfluidic channel is flat, the entity/surface interaction generally takes place at the beginning of the main channel (4) and if a target entity is not captured at the upstream of the channel, the probability of it being captured later on drastically decreases (Figure 4) . This is mainly due to the fact that the fluid flow (1 ) becomes uniform and the entities follow distinct streamlines which, at low Reynolds number (< < 1 ) , do not coincide with the pillars (5) in the microchannel. I n order to keep the entity/surface interaction probability at a high value throughout the whole channel, the chaotic trajectories of the biological entities (1 ) at the upstream of the channel should be maintained.

I n the claimed devices, the channel is formed as the combination of flipping ellipse segments (1 1 ) (successive chords facing opposite directions) connected to each other, such that the starting edge of the successive ellipse segment is located at the halfway of the chord of the preceding ellipse segment (Figure 5) .

I n this proposed invention, it is claimed to maintain the chaotic trajectories of the biological entities throughout the whole channel with a device in which the attack angle between the biological entity trajectory and the droplet shaped pillars continuously changes throughout the channel, while the pillar shape and distribution are kept constant. In a particular embodiment, exemplary computer simulation of the flow inside two attached ellipse segments having an example array of pillars shows the biological entity trajectory inside this formed microfluidic channel area with an inlet and an outlet. Biological entities, such as cells, enters at the inlet and follows the fluid streamlines which orients the same biological entities towards the pillar and/or microfluidic channel walls. Due to the bending of the flow inside the ellipse segments, biological entities collide with the pillar at different attack angles (0°-180°) (Figure 6).

In a particular embodiment, the ellipse segment (11) is a halve circle. In a particular embodiment, three ellipse segments (11) form the main channel (4). In a particular embodiment, five ellipse segments (11) form the main channel (4). In a particular embodiment, there are eight ellipse segments and fourth and fifth ellipse segments (11) are connected to each other through a straight microchannel (4) comprising the same pillar (5) pattern, all of the eight ellipse segments (11) and the straight microchannel (4) forming the channel.

REFERENCES

• Harb W., Fan A., Tran T., Danila D.C., Keys D., Schwartz M., lonescu-Zanetti C., Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay, Translational Oncology Vol.6, No.5, 2013.

• Winer-Jones J.P., Vahidi B., Arquilevich N., Fang C., Ferguson S., Harkins D., Hill C., Klem E., Pagano P.C., Peasley C., Romero J., Shartle R., Vasko R.C., Strauss W.M., Dempsey P.W., Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell, PLOSONE, Vol 9, Issue 1, e86717, 2014.

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2006/0160243A1.

• Skelley A., Smirnov D., Dong Y., Merdek K.D., Sprott K., Carney W., Jiang C., Huang R., Lupascu I., (2014). United States Patent No. US 2014/0154703A1.

• Fuchs M., Toner M., (2007). United States Patent No. US 2007/0026417A1.