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Development of SERS-based multiplex immunoassay platforms for accurate prostate cancer diagnosis

Title
Development of SERS-based multiplex immunoassay platforms for accurate prostate cancer diagnosis
Other Titles
표면 증강 라만 분광법 기반 정확한 전립선 암 진단 다중 면역분석 플랫폼 개발
Author
Cheng Ziyi
Advisor(s)
Choo Jaebum
Issue Date
2019-02
Publisher
한양대학교
Degree
Doctor
Abstract
Surface-enhanced Raman scattering (SERS) spectroscopy provides a highly sensitive molecular detection method in extensive areas including polymer science, materials science, biochemistry and biosensing, catalysis, and electrochemistry. In recent years, SERS combined with nanotechnology is considered a promising technique in the biosensing field. SERS-based assay has shown great potential as a multiplexed detection method in clinical diagnosis of cancer. This is because simultaneous detection of multiple biomarkers plays an important role in the accurate diagnosis of cancer. The approach of using SERS-based multiplexed immunoassay will provide another insight into the accurate diagnosis of cancer. Microfluidics technology is also extensively used in biochemical research. This is because it can realize a miniaturized, high-throughput, and automated assay platform. The combination of SERS with microfluidic systems has many advantages over the conventional macroscale systems. For instance, the capability to operate in a continuous flow regime as well as to generate homogeneous mixing conditions affords highly accurate and automatic SERS-based quantitative analysis. Furthermore, the combination of SERS and microfluidic devices provide an ideal mechanism for achieving sensitive detection, reproducible measurements, and spatially defined detection areas. An added benefit of such integrated systems for bio-analytics is the ability to take measurements using minimal sample volumes and low analyte concentrations. In the real clinical diagnosis scene, individual detection of one biomarker is not enough to accurately diagnose a target disease. In this work, we demonstrated an accurate diagnosis of a specific cancer based on simultaneous detection of two specific biomarkers in clinical serum. It is important for early diagnosis and treatment of cancer. Herein, a SERS-based immunoassay, using magnetic beads together with SERS nanotags, was developed for the determination of free-to-total (f/t) prostate-specific antigen (PSA) ratio to improve the diagnostic performance of prostate cancer. To assess the clinical capability of our method, SERS-based assays for the simultaneous detection of dual PSA markers, free PSA (f-PSA) and complexed PSA (c-PSA), were performed for clinical samples in the total PSA gray zone: 4.0 - 10.0 ng/mL. Our assay results for f/t PSA ratio showed a good linear correlation with those measured using the conventional electrochemiluminescence (ECL) system installed in the clinical laboratory of a University Hospital. In addition, the simultaneous assay provided better precision than parallel assays for the detection of f-PSA and c-PSA in 13 clinical serum samples. Therefore, our SERS-based assay for simultaneous detection of dual PSA markers in clinical fluids has a strong potential for application in the accurate diagnosis of prostate cancer. As we discussed above, to combine the remarkable advantages of microfluidic devices with SERS-based immunoassay, a novel wash-free magnetic immunoassay microdroplet sensor was designed for the detection of prostate-specific antigen (PSA) using surface-enhanced Raman scattering (SERS). The free and bound SERS tags were separated by splitting the droplets into two smaller parts with a magnetic bar embedded in the droplet microfluidic device. The presence of PSA targets leads more SERS tags to immunocomplex in one droplet so that fewer SERS tags remain in another supernatant solution droplet. Thus, SERS signal measurement enables the quantitative evaluation of specific PSA markers. This approach provides a sensitive and rapid assay method that is applicable for PSA cancer markers detection in human serum without any washing step. Specifically, SERS signals were measured at 174 droplets per minute and averaged for quantitative evaluation of PSA. The limit of detection (LOD) determined by our SERS-based microdroplet chip was estimated to 0.1 ng/mL, which is below the clinical cut-off point for diagnosing prostate cancer. In addition, the entire assay can be carried out automatically, only a minimal amount of sample is needed. Accordingly, the approach is expected to be useful as a potential clinical tool for the early diagnosis of prostate cancer. To further improve the diagnosis accuracy, we also report a novel surface-enhanced Raman scattering (SERS)-based microfluidic device for the simultaneous detection of f-PSA and t-PSA markers. A fully automatic droplet-based microfluidic platform for the rapid and sensitive detection of f-PSA and t-PSA was designed. Magnetic immunocomplexes were aligned on one side of the channel using a permanent magnet embedded in the microfluidic device, and parent microdroplets containing magnetic immunocomplexes and supernatant solutions were split into two smaller daughter droplets at the Y-shaped junction of the channel. Then, Raman signals of sequential droplets including supernatant solutions were measured for the quantitative analysis of the PSA markers. Two parallel microfluidic channels were designed and fabricated for the simultaneous detection of f-PSA and t-PSA. Our results showed a good linearity range for both PSA markers in the range from 0.05 to 100 ng/mL. The limits of detection were estimated to be below 0.1 ng/mL for both the f-PSA and t-PSA. This SERS-based assay in a microfluidic channel was completed in 10 min without any manual incubation and washing steps. Our method is a very promising clinical tool for PSA-based screening test of prostate cancer.
URI
http://dcollection.hanyang.ac.kr/common/orgView/000000107763http://repository.hanyang.ac.kr/handle/20.500.11754/100405
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Theses (Ph.D.)
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