Investigation on the isolation and detection of circulating tumor cells based on microfluidics and surface-enhanced Raman scattering method

Abstract

Cancer is a leading cause of death and is an important barrier to increase life
expectancy in every country of the world. Besides, two thirds of cancer death
occurred in less developed countries because of delayed diagnosis and less
accessible treatment. The delayed diagnosis would lead to increased death and
disability from cancer. Therefore, it is essential to develop a technology for
cancer detection with universality and less cost. The common imaging technologies, such as Magnetic Resonance Imaging (MRI), Computed
Tomography (CT) and Positron Emission Tomography (PET), can be used for
cancer early screening. After early screening, people who have symptoms and
signs consistent with cancer, require further identification of cancer by
pathological diagnoses. The traditional invasive biopsy would cause
psychological burden to patients and is limited by sample collection in deep
tumor and sampling bias. It also has risk of tumor metastasis. In recent decades,
liquid biopsy technique has developed quickly and attracted more and more
attention. Compared to invasive biopsy, liquid biopsy is noninvasive, cheaper,
simple for sample collection with minimum risk. The sensitivity and specificity
of liquid biopsy technique has also been improved remarkably due to the
continuous innovation in molecular biology technology. Liquid biopsy
generally includes the detection of circulating tumor cells (CTCs), circulating
tumor-derived exosomes and circulating tumor nucleic acids. Circulating tumor
cells are tumor cells that shed from solid tumor and circulate in the blood.
Circulating tumor-derived exosomes are vesicles that secret from tumor cells
containing some ribonucleic acids (RNA) and protein. Circulating tumor
nucleic acids are nucleic acids shedding from tumor cells or released by
apoptotic cells. Compared to circulating tumor-derived exosomes and
circulating tumor nucleic acids, circulating tumor cells are integrated cells
carrying more complete information about tumor. Circulating tumor cell (CTC)
detection as a burgeoning detection strategy can identify the tumor lesion in the
early stage, and facilitate the understanding of tumorigenesis, tumor
progression, metastasis, and drug-resistance. As so far, many technologies for
CTC detection have been developed. Generally, CTC detection can be divided
into two stages: first is isolation and enrichment of CTCs and second is
downstream analysis of CTCs. There are three main challenges exciting in
CTC detection: small number of CTCs, complex blood background, and
diversified typing of CTCs. To overcome these difficulties, microfluidic
method has been applied to improve CTC isolation combined with Raman
fingerprint spectra and surface-enhanced Raman scattering (SERS) method to
distinguish CTCs from other blood components. Current microfluidic method
cannot separate CTCs completely from blood, which requires further
downstream analysis to distinguish CTCs from remaining blood cells. Besides,
SERS method might be interfered by complex blood components, which would
reduce detection sensitivity of SERS method. Therefore, blood samples require
pretreatment to reduce the interference of blood cells for SERS detection and
improve repeatability and reliability of detection results. In this thesis, SPIONPEI@Au based SERS biological probe and B-TiO2 based SERS biological
probe were prepared for direct detection of CTCs in the blood. To improve the
detection sensitivity and accuracy, microfluidic method was combined for
blood pretreatment before SERS detection. The specific research contents
mainly include the following three parts:
1. Superparamagnetic iron oxide nanoparticles with poly(ethyleneimine) coated with gold nanoparticles (SPION-PEI@Au) were synthesized according to the previous work of the research group. In this work, SPIONPEI@Au based SERS biological probe was applied on CTC detection of clinical blood samples. The SPION@Au-MBA-rBSA-FA SERS biological probe consisted of four parts: SPION-PEI@Au composite nanoparticles as SERS substrate, 4-mercaptobenzoic acid (MBA) as Raman reporter, reduced bovine serum albumin and folic acid (rBSA-FA) to recognize folate receptor (FR) on cancer cell membrane. In this work, 32 clinical blood samples from cancer tumor and 3 clinical blood samples from
healthy people were detected directly by SPION@Au-MBA-rBSA-FA SERS biological probe.
2. Black TiO2 (B-TiO2) nanoparticles were used as SERS substrate. B-TiO2
has advantages of low cost, high spectral stability and reproducibility, strong anti-interference ability, and selective SERS enhancement to target molecules. The synthesized B-TiO2 showed good SERS enhancement effect and the LOD of the AR molecule on B-TiO2 can reach to 5×10-8 M. The B-TiO2-AR-PEG-FA biological probe consisted of four layers. The innermost layer was B-TiO2 nanoparticles with crystal core and amorphous shell structure. The second layer was alizarin red (AR) molecule which was responsible for providing Raman spectral signal. The third layer was a thin NH2-PEG2000-COOH layer which was used to improve the dispersion of biological probe and to provide binding sites of folic acid (FA) and thus to increase FA grafting rate. The outermost layer was FA molecule. FA was used to specifically recognize cancer cells by folate receptor (FR) on cancer cell membrane. The research results showed that this B-TiO2 based SRES biological probe has good specificity and detection accuracy with obvious Raman signal. It can distinguish positive FR-expressing cancer cells (MCF7) from lower FR-expressing cells (A549 and Raw264.7).
3. Because of complex blood components, the sensitivity and stability of SERS biological probe would be attenuated without proper blood pretreatment. To improve the performance of SERS biological probe, microfluidic method was added to isolate CTCs before SERS detection. In this strategy, the relatively low specificity and isolation purity of microfilter could be solved by integrating with highly sensitive and highly specific SERS spectra detection, while the microfilter could reduce the interference of blood background to SERS detection. Besides, SERS-fluorescence dualmodal in situ imaging method proved that this strategy has high specificity with detection limit of 2 cancer cells per milliliter in rabbit blood. Besides, the operation process was simple and high-speed, with detection time less than 1.5 hours. This strategy has also been applied to CTC detection of
clinical blood samples and has detected CTCs from blood successfully.

These results illustrated that in addition to noble metal nanoparticles, semiconductor nanoparticles can also be used for SERS detection with good SERS enhancement effect. Although SERS method has good sensitivity in CTC detection, the performance of SERS method would be affected by complex components of blood and improper blood treatment. Microfluidic method was combined to remove the interference of blood cells and to improve the repeatability and reliability of SERS detection. The combination of microfluidic method and SERS detection method could complement their own shortcomings and thus to improve the detection efficiency. This thesis demonstrated that the combination of microfluidic method and SERS detection method could open new paths for liquid biopsy.

Date of Award	Jul 2023
Original language	English
Awarding Institution	University of Nottingham
Supervisor	Yong Ren (Supervisor)