Scope and Objectives

Our aim is to build a prototype of an affordable technology platform to detect CTCs in solid cancers. The detectable technology is divided into two separate parts, a microfluidic device to enrich CTCs and an optical sensor system. The CTC-enriched blood, captured with the help of microfluidic chip, will be passed over a sensitive and highly selective optical sensor probe. The detection is useful for prognosticating the stage of the cancer and metastasis. The major scope of the validated technology will lie in translating CTC detection from research labs to the clinics and thus will be suitable for use in hospitals all over the country.

Deliverables

The deliverable will be a marketable prototype of an optical fiber sensor integrated with microcontroller unit and flow cell system. Automatic LED stabilization as well as least pre-processing of minimal sample volume will be taken into account. Built-in probe fitting mechanism will be developed with minimum switches for user friendly operations. The LED display will provide an automatic and visual read-out mechanism to determine the concentration of analytes effectively. The main beneficiary will be the healthcare sector, particularly pathology labs, health insurance companies, etc.

Scientific Output

The sensor has two components: Microfluidics system for CTCs enrichment and optical probe for CTC detection.

1. Development of microfluidic device and centrifugal prototype for CTC enrichment:
Our goal is to get rid of all RBCs, all platelets, and most of the WBCs from this device. We expect WBCs and CTCs to be trapped by the pillars, while the RBCs and platelets should flow into the waste outlet. Once all RBCs and platelets are removed from the sample, we will flow a buffer solution from the outlet (i.e. reverse flow) and collect the captured WBCs and CTCs from the inlet. In the later versions of the designs, we will coat the pillars with antibodies to capture WBCs (negative enrichment), such that only CTCs will be collected by the reverse-flow. It should be noted that the aim of the current design is to simply enrich the population of CTCs in blood by negative depletion of WBCs. We also explored the development of a portable centrifugal prototype to pump samples into the chip. Normally samples are pumped using a syringe pump. We wished to rotate the chip and use centrifugal force to push the liquid in it. The advantage of this approach is that the prototype could be operated with 5V from a USB port of any laptop and eliminates the requirement of continuous pumping of sample. 

2. Optical probe for CTC detection:
The enriched sample of rare circulating tumor cells from the microfluidics system is detected by an optical system. For this system, the primary sensing element will be a suitably functionalized optical fiber or an optical waveguide. The waveguide/ fiber will be U-shaped in order to increase the strength of the evanescent power, giving rise to a highly sensitive system. The fiber/waveguide will be coated with gold nanoparticles (GNP) or polyaniline (PANI) which is further functionalized with a receptor.

• The cancerous cells express a set of biomarkers on its surface different from non-cancerous cells, which can be employed for detection of these cells. An extensive literature survey was carried out for the selection of the appropriate biomarker for detection of these cells. The suitable surface marker for CTC detection has been identified.

• We started experiments with GNP coated U-bend optical fiber which were prepared according to optimized protocol in lab.  The antibodies and aptamers targeting the extracellular domains of selected surface markers on cancer cells were then immobilized on the probe. The protocol for their immobilization on U-bend optical fiber has been optimized. We confirmed there immobilization on the probe by using fluorescence microscopy.

• Once the system was ready, we tried detecting MCF-7 cells by it.  So far, our system was able to detect approx.  10 cells suspended in 200 μL of PBS. Thereafter, we performed experiments with HeLa and RB-1 cells and the system was also able to detect them. 

Results and outcome till date

Proposed Milestones :Status
1.  Obtain aptamers and antibodies commercially: Primary Antibodies and aptamers have been purchased.
2. Standardize immobilization protocols based on the earlier work. Develop an immunoassay protocol for immobilization of biomolecules: The Gold Nanoparticles coated probe has been made based on lab protocol. For antibody and aptamer immobilization protocols have been optimized.
3. Carry out initial experiments on Bench top platform as a proof of concept for detection of analyte using both GNP as well as PANI in distilled water/buffer.: The experiments with GNP probe using MDA-MB-231, MCF-7, HeLa, and RB-1cells in PBS were started. The system was able to detect these cells. The protocol with PANI yet to be optimized.
4.  Development of CTC enrichment chip: Design of the first version of the chip is completed. Fabrication of master and PDMS chip is completed; we have completed micro-bead experiment. Currently, experiments with buffy coat and cancer cell line MCF-7 are going on.
5. Development of centrifugal prototype: We have developed initial version of prototype and moving towards final design of the same.

Societal benefit and impact anticipated

Detection and capture of CTCs from blood samples of cancer patients is of immense importance in cancer staging, clinical decision making, and also for evaluating the metastatic spread of cancer. The device we are developing for CTC detection will be a portable, user-friendly and reliable which will be suitable for operating over diverse regions in our country. We are trying to develop a very specific and highly sensitive system with least processing steps and thus will be fast. The system can be used by variety of users like: Healthcare sector including various hospitals (pathology labs, oncology departments, as well as emergency departments for rapid analysis), private pathology and diagnostic labs, Mobile clinics that can reach places in and around the country to provide healthcare management at rural level. There is a huge opportunity to use the chip by biotech companies for isolating CTCs. The sensor can also be used by educational institutes for acquainting the students with new detection technologies.

Next steps

Develop immobilization protocol to eliminate non-specific binding from proteins in blood sample
Experiments on U-bent probe for detection of analyte in real samples (not buffer).
Design and development of electronics that includes stabilization of LED’s, Microcontroller and detector unit
Development of instrument and testing using simulated samples. Design iterations based on feedback.
Optimization of chemical binding of WBC to the pillar in microfluidic chip.
Development of centrifugal prototype.
Experiments with spiked cancer cell in whole blood for CTC enrichment using microfluidic device
Experiments with clinical CTC sample, in order to enrich CTC from whole blood.

Publications and reports

“Towards a portable radial pillar device (RAPID) for clog-free continuous cell separation”, Ninad D. Mehendale, Shilpi Pandey, Oshin Sharma, Misal Choudhary and Debjani Paul, Microfluidic 2018: New Technologies and Applications in Biology, Biochemistry and Single-Cell Analysis organizing by EMBL, Germany from 15th – 17th July, 2018.

Patents

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Scholars and Project Staff

• Rajshree Gupta (PhD Student)

• Kavya Subramanian (Junior Research Fellow)

• Jasleen Chhabra (Senior Research Fellow)

Challenges faced

• The immobilization of aptamers on the GNP coated U-bend optical probe was challenging. This was first attempt of aptamer immobilization on the optical probe.

• Due to deformability of MCF-7 cells, optimizing the pillar gap for cells capturing is very difficult.