Molecular Sensing and Imaging of Human Disease Cells and Their Responses to Biochemical Stimuli

The overall goal of this dissertation is to develop noninvasive imaging techniques that allow us not only to detect diseased cells but also to study the molecular mechanisms underlying these diseases. Atomic force microscopy and Raman spectroscopy are applied to measure cellular mechanical properties (e.g. Young’s Modulus, adhesion force) and biochemical composition of living cancerous vs. healthy (A549 vs. SAEC) human lung epithelial cells. These biomechanical and biochemical properties can be utilized to differentiate between cancerous A549 and healthy SAEC human lung epithelial cells. Furthermore, different cellular responses to anticancer drug doxorubicin (DOX) treatment are also observed. Using AFM and Raman spectroscopy, we can quantitatively measure biophysical properties of different cells, as complementary parameters to other properties (e.g. gene and protein expression), helping identify the states of diseased cells. Another major task of this dissertation is to develop noninvasive imaging techniques to detect cancer biomarker epidermal growth factor receptor (EGFR) at single cell level using advanced instrumentation. We first synthesized a gold nanorod (AuNR)-based nanoprobe for single-cell imaging of EGFR using surface-enhance Raman spectroscopy (SERS). SERS is able to quantitatively measure the EGFR expression level in different breast cancer cell lines and map the cellular distribution of EGFR in single cells. Moreover, SERS, as a noninvasive imaging technique, is able to monitor the process of nanoparticle uptake by single cell. Due to the diffraction limit of optical microscopy, SERS is unable to provide nanoscale imaging resolution. We then applied an AFM-based simultaneous Topography and RECognition (TREC) imaging technique to image EGFR with nanoscale resolution. TREC is first validated on mica surface and then successfully utilized to map the EGFR distribution in fixed and living breast cancer cells at single molecule level. In addition, we have explored the potential of a gadolinium-gold (Gd-Au) composite nanomaterial as a dual functional (MRI-SERS) imaging probe. Using this previous reported MRI contrast agent, we successfully apply SERS function in the detection of EGFR in three cancer cell lines. The last part of the dissertation is to study fat-responsive G protein-coupled receptor 120 (GPR120), and its interaction with linoleic acid (LA). We have synthesized a dual functional composite nanoparticle for SERS-fluorescence bimodal imaging of GRP120 in living HEK293 cells. By SERS-fluorescence imaging, we are able to locate GPR120 distribution in single cells. Moreover, we have observed a dose-dependent GPR120 response to LA treatments using SERS. This work demonstrates the potential to use SERS-fluorescence bimodal imaging technique for real-time detection of the interaction between fatty acids and their receptors (e.g. GPR120, CD36)

Use of Surface-Enhanced Raman Scattering (SERS) Probes to Detect Fatty Acid Receptor Activity in a Microfluidic Device

In this study, 4-mercaptobenzoic acid (MBA)-Au nanorods conjugated with a GPR120 antibody were developed as a highly sensitive surface-enhanced Raman spectroscopy (SERS) probe, and were applied to detect the interaction of fatty acids (FA) and their cognate receptor, GPR120, on the surface of human embryonic kidney cells (HEK293-GPRR120) cultured in a polydimethylsiloxane (PDMS) microfluidic device. Importantly, the two dominant characteristic SERS peaks of the Raman reporter molecule MBA, 1078 cm−1 and 1581 cm−1, do not overlap with the main Raman peaks from the PDMS substrate when the appropriate spectral scanning range is selected, which effectively avoided the interference from the PDMS background signals. The proposed microfluidic device consisted of two parts, that is, the concentration gradient generator (CGG) and the cell culture well array. The CGG part was fabricated to deliver five concentrations of FA simultaneously. A high aspect ratio well structure was designed to address the problem of HEK cells vulnerable to shear flow. The results showed a positive correlation between the SERS peak intensity and the FA concentrations. This work, for the first time, achieved the simultaneous monitoring of the Raman spectra of cells and the responses of the receptor in the cells upon the addition of fatty acid. The development of this method also provides a platform for the monitoring of cell membrane receptors on single-cell analysis using SERS in a PDMS-based microfluidic device

Microfluidic Chip for Non-Invasive Analysis of Tumor Cells Interaction with Anti-Cancer Drug Doxorubicin by AFM and Raman Spectroscopy

Raman spectroscopy has been playing an increasingly significant role for cell classification. Here, we introduce a novel microfluidic chip for non-invasive Raman cell natural fingerprint collection. Traditional Raman spectroscopy measurement of the cells grown in a Polydimethylsiloxane (PDMS) based microfluidic device suffers from the background noise from the substrate materials of PDMS when intended to apply as an in vitro cell assay. To overcome this disadvantage, the current device is designed with a middle layer of PDMS layer sandwiched by two MgF2slides which minimize the PDMS background signal in Raman measurement. Three cancer cell lines, including a human lung cancer cell A549, and human breast cancer cell lines MDA-MB-231 and MDA-MB-231/BRMS1, were cultured in this microdevice separately for a period of three days to evaluate the biocompatibility of the microfluidic system. In addition, atomic force microscopy (AFM) was used to measure the Young\u27s modulus and adhesion force of cancer cells at single cell level. The AFM results indicated that our microchannel environment did not seem to alter the cell biomechanical properties. The biochemical responses of cancer cells exposed to anti-cancer drug doxorubicin (DOX) up to 24 h were assessed by Raman spectroscopy. Principal component analysis over the Raman spectra indicated that cancer cells untreated and treated with DOX can be distinguished. This PDMS microfluidic device offers a non-invasive and reusable tool for in vitro Raman measurement of living cells, and can be potentially applied for anti-cancer drug screening

Artificial intelligence : A powerful paradigm for scientific research

Y Artificial intelligence (AI) coupled with promising machine learning (ML) techniques well known from computer science is broadly affecting many aspects of various fields including science and technology, industry, and even our day-to-day life. The ML techniques have been developed to analyze high-throughput data with a view to obtaining useful insights, categorizing, predicting, and making evidence-based decisions in novel ways, which will promote the growth of novel applications and fuel the sustainable booming of AI. This paper undertakes a comprehensive survey on the development and application of AI in different aspects of fundamental sciences, including information science, mathematics, medical science, materials science, geoscience, life science, physics, and chemistry. The challenges that each discipline of science meets, and the potentials of AI techniques to handle these challenges, are discussed in detail. Moreover, we shed light on new research trends entailing the integration of AI into each scientific discipline. The aim of this paper is to provide a broad research guideline on fundamental sciences with potential infusion of AI, to help motivate researchers to deeply understand the state-of-the-art applications of AI-based fundamental sciences, and thereby to help promote the continuous development of these fundamental sciences.Peer reviewe

Microfluidic Chip for Single Cell Analysis of Tumor Cells Interaction with Anti-Cancer Drug Doxorubicin by AFM and Raman Microspectroscopy

The advantages of Raman spectroscopy include high efficient, high specific and label free, and therefore, the method is also eligible to live cells. In this study, a important technical innovation is reported here towards Raman cell identification. A microfluidic chip made of MgF2 is introduced for Raman cell natural spectral fingerprint collection. The cancer cells were cultured in device for a period of 3 days to validate the biocompatibility of the microfluidic system. In addition, Both AFM and Raman spectroscopy were applied to detect the responses of A549, 231 and 231-B cells to DOX exposure (24h) at the single cell level. Principal component analysis (PCA) and atomic force microscope (AFM) results basically agree with our previously published results tested by traditional method. The Microfluidic cell culture platform is straightforward, reusable and it has not been reported in the literature. This microfluidic device system provide a new platform for Raman cancer cell research and clinic cancer diagnostic

Spatial-Temporal Evolution Relationship between Water Systems and Historical Settlement Sites Based on Quantitative Analysis: A Case Study of Hankou in Wuhan, China (1635–1949)

When deciding on and creating their own settlement environment, humans’ relationships with water resources have evolved. From the earliest times when they observed water and learned about its characteristics and laws to create artificial rivers, to the gradual development and use of water resources to create water plants and pumping stations, to the management of water resources to set up customs and dams to prevent and manage water hazards.To lay the groundwork for more sustainable development of the relationship between humans and water in the city, it is important to understand and summarize this state of change. Wuhan, known as the “City of a Thousand Lakes”, is a typical case of studying the traditional relationship between Chinese people and water, and can better provide modern cities with the value of historical experience in sustainable development. Therefore, this study takes the Hankou town of Wuhan from 1635 to 1949 as the research object, uses historical maps and written materials as data sources, and creates a database of historical information based on the water system of Hankou and the sites of artificial settlements such as buildings and streets. It takes quantitative analysis and map visualization techniques of the GIS platform from the perspective of quantitative historical research. Firstly, it creates a database of historical information based on the water system of Hankou and the sites of artificial settlements such as buildings and streets. Secondly, it gives the quantitation about the human–water relationship in Hankou by applying the spatial analysis methods of buffer analysis. The study’s findings demonstrate that from 1635 to 1864 there were an increasing number of artificial settlement sites that were distributed along the water system, keeping a reasonable distance from the water; from 1684 to 1905, people constructed dikes to prevent flooding, which resulted in an increase in urban space; and from 1905 to 1949, the development of Hankou shifted toward the Hanjiang River and the Yangtze River. The procedure shows a change in the relationship between avoiding water and subsequently managing water and using water. The results of the study indicate the following: (1) Water is essential for the environment of human settlements. (2) Human activities have an impact on the structure of water systems. (3) There is a high degree of coupling between the Hankou urban water system and the sites of artificial settlements. It proves that the relationship between humans and water is very close in the process of modern urbanization in Hankou. In building a traditional habitat environment to regulate water, it is consistent with the ancient Chinese concepts of “harmony between man and nature” and “the best place to live is close to water conservancy but also avoid flood.” This paper is helpful for re-examining and establishing the harmonious relationship between humans and water to encourage sustainable urban growth and reshape the urban spatial environment with Chinese characteristics. It also provides a method based on quantitative analysis for studying the evolution history of urban settlement environments

Selective Detection of RGD-Integrin Binding in Cancer Cells Using Tip Enhanced Raman Scattering Microscopy

Ligand–receptor interactions play important roles in many biological processes. Cyclic arginine–glycine–aspartic acid (RGD) containing peptides are known to mimic the binding domain of extracellular matrix protein fibronectin and selectively bind to a subset of integrin receptors. Here we report the tip enhanced Raman scattering (TERS) detection of RGD-functionalized nanoparticles bound to integrins produces a Raman scattering signal specific to the bound protein. These results demonstrate that this method can detect and differentiate between two different integrins (α5β1 and αvβ3) bound to RGD-conjugated gold nanoparticles both on surfaces and in a cancer cell membrane. In situ measurements of RGD nanoparticles bound to purified α5β1 and αvβ3 receptors attached to a glass surface provide reference spectra for a multivariate regression model. The TERS spectra observed from nanoparticles bound to cell membranes are analyzed using this regression model and the identity of the receptor can be determined. The ability to distinguish between receptors in the cell membrane provides a new tool to chemically characterize ligand–receptor recognition at molecular level and provide chemical perspective on the molecular recognition of membrane receptors

In vitro Biophysical, Microspectroscopic and Cytotoxic Evaluation of Metastatic and Non-metastatic Cancer Cells in Response to Anti-cancer Drug

The Breast Cancer Metastasis Suppressor 1 (BRMS1) is a nucleo-cytoplasmic protein that suppresses cancer metastasis without affecting the growth of the primary tumor. Previous work has shown that it decreases the expression of protein mediators involved in chemoresistance. This study measured the biomechanical and biochemical changes in BRMS1 expression and the responses of BRMS1 to drug treatments on cancer cells in vitro. The results show that BRMS1 expression affects biomechanical properties by decreasing the Young\u27s modulus and adhesion force of breast cancer cells after doxorubicin (DOX) exposure. Raman spectral bands corresponding to DNA/RNA, lipids and proteins were similar for all cells after DOX treatment. The expression of cytokines were similar for cancer cells after DOX exposure, although BRMS1 expression had different effects on the secretion of cytokines for breast cancer cells. The absence of significant changes on apoptosis, reactive oxygen species (ROS) expression and cell viability after BRMS1 expression shows that BRMS1 has little effect on cellular chemoresistance. Analyzing cancer protein expression is critical in evaluating therapeutics. Our study may provide evidence of the benefit of metastatic suppressor expression before chemotherapy

Experimental Investigation of Solidification in the Cast Mold with a Consumable Cooler Introduced Inside

The microstructure is of great significance for the stability and mechanical performance of the cast slab. Recently, an innovative technology of feeding a consumable cooler into the mold has been proposed to improve the internal quality of castings. But the mechanism is not clear. In the present work, a water-cooled transparent laboratory equipment was set up and solidification of NH4Cl-70%H2O solution was studied to observe the in-situ growth and sedimentation of crystals. The experiments were conducted with and without adding a consumable cooler. Morphology variation of the solidification structure was visualized and temperature distribution during the process was recorded. Results show that introduction of the consumable cooler significantly reduces the temperature of the central zone. Melting of the consumable cooler can supply a large quantity of equiaxed crystals, which prevent the growth of columnar dendrites and thereby promote columnar to equiaxed transition (CET). Moreover, the enhanced convection shows an effect of grain refinement