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Cytokines are small-molecule signaling proteins involved in cell-cell regulation. The detection of low-abundance cytokines is challenging since the currently available techniques are limited by sensitivity and are time-consuming. Nanopore sensing is an emerging technique in nanotechnology that is catalyzing key breakthroughs in many areas, including the analysis and study of proteins at the single-molecule level. Solid-state nanopore sensing has the advantage of analyzing small copy numbers of biomolecules, such as DNA, with high throughput. However, protein detection using nanopores is still in in infancy because the mechanisms of native protein translocation inside the solid-state nanopore are highly complicated. The goal of this project is to develop a novel solid-state nanopore device for identification and quantification of cancer cytokines directly from cell culture. Vascular endothelial growth factor (VEGF) is chosen as a model cytokine due to its high abundance in cancerous tissue, and its well-characterized molecular structure. Firstly, we used a nanopore sensor to monitor individual VEGF proteins in solution while simultaneously obtaining tertiary and quaternary structural information. Next, we used the translocation signature to identify VEGF secreted directly from the culture media of the breast cancer cell line. A series of DNA and RNA aptamers was screened to selectively bind to secreted VEGF, enhancing the detection rate and creating a unique translocation signature for easy protein discrimination. Finally, we integrated the nanopore with a hard microfluidic device designed to facilitate the on-chip sample preparation prior to nanopore sensing. This nanopore-microfluidic device may allow scientists and clinicians to directly detect biomarkers secreted from a small population of cultured cells, which would revolutionize cancer diagnostics and prognostics. / 2020-10-22T00:00:00Z

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15407号 / 工博第3286号 / 新制||工||1495(附属図書館) / 27885 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 白川 昌宏 / 学位規則第4条第1項該当

Proteins are of primary importance to the structure and function of all living cells. Study of proteins relies on the ability to separate a complex mixture so that individual proteins can be more easily processed by other techniques. Since protein samples often exist at low concentration in a small volume, the trend in chemical analysis is toward micro total analysis systems (µTAS) or lab-on-a-chip devices. Among µTAS separation methods, the relatively new electric field gradient focusing (EFGF) technique has shown potential. It focuses and separates analytes based on their electrophoretic migration in an opposing hydrodynamic flow. The detection principles that are compatible with µTAS separation may not always scale down. This thesis represents the development of laser-induced two-photon fluorescence detection on a microchip separation device. This detection is based on excitation of native fluorescence of aromatic amino acids by simultaneous absorption of two photons. First, a compact two-photon prototype detector was investigated. Its sensitivity was improved after discovery of the source of the background and subsequent reduction of background levels. Simple CE separation on a square capillary was coupled to this detector to demonstrate its ability for micro-scale detection. However, this detector did not provide a way to view the location illuminated by a laser and was difficult to use for on-microchip detection. A two-photon microscope was constructed on the frame of a commercial Olympus microscope to solve this problem. The eyepiece of this microscope enabled viewing of the detection volume, and the removal of a glass compensator from the trinocular head allowed for UV detection. This detection system was carefully aligned and optimized before coupling to microchip CE. Two microchip substrates including poly (methyl methacrylate) (PMMA) and glass, and two chip layouts were explored for their compatibilities with the microscope detector. It was found that the PMMA chip with conventional chip layout was not suitable for two-photon detection; therefore, a novel chip layout on PMMA was designed. Through testing the new design, it was concluded that precise focusing of the laser was essential to successful detection on microchips. Although the precise focusing of the laser inside microchip channels was not achieved completely in the limited research period, it is believed that this new design should be an appropriate solution to coupling PMMA chips with the two-photon microscope. Finally, glass chips were employed to successfully demonstrate the detection of amino acids.

Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.   In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.   Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications. / QC 20100719

Fluorescence-based assays have gained an ever-increasing popularity in life sciences. One of these rapidly emerging techniques is Protein Induced Fluorescence Enhancement (PIFE). Traditional explanations of PIFE focused exclusively on the role of the protein and largely neglected the role of the mediator DNA. In the same time, the existing models of PIFE were denying its exactly opposite effect. In the first part of the current dissertation we focus on a better understanding of PIFE, stimulated by the direct observation of its opposite effect, Induced Fluorescence Enhancement Quenching (PIFQ). This study offered us the leverage for obtaining on-demand fluorescence modulation in cyanine dyes. The following two chapters harvest this control over fluorescence modulation to generate two biotechnology applications: a sensitive potassium sensor with embedded fluorescent transducer, and a simple protocol for the fluorescent detection of His-tagged proteins. In the last part, a variety of fluorescence tools including Förster resonance energy transfer, fluorescence enhancement, and fluorescence quenching are employed for a much more complex task; to demystify the behavior of the human Maturation of Okazaki Fragments (MOF) machinery. First, we reconstituted the human MOF reaction and showed that it behaves considerably different than its well-established yeast homolog. Subsequently, our toolbox of fluorescence-based assays was used to pinpoint the kinetics and dynamics that lead to this unexpected MOF behavior.

This thesis describes a new approach to biomolecular analysis, called the volume-amplified magnetic nanobead detection assay (VAM-DNA). It is a sensitive, specific magnetic bioassay that offers a potential platform for the development of low-cost, easy-to-use diagnostic devices. The VAM-NDA consists of three basic steps: biomolecular target recognition, enzymatic amplification of the probe-target complex using the rolling circle amplification (RCA) technique, and addition of target complementary probe-tagged magnetic nanobeads which exhibit Brownian relaxation behavior. Target detection is demonstrated by measuring the frequency-dependent complex magnetization of the magnetic beads. The binding of the RCA products (target DNA-sequence coils) to the bead surface causes a dramatic increase in the bead size, corresponding essentially to the size of the DNA coil (typically around one micrometer). This causes a decrease in the Brownian relaxation frequency, since it is inversely proportional to the hydrodynamic size of the beads. The concentration of the DNA coils is monitored by measuring the decrease in amplitude of the Brownian relaxation peaks of free beads. The parameters oligonucleotide surface coverage, bead concentration, bead size and RCA times were investigated in this thesis to characterize features of the assay. It was found that all of these parameters affect the outcome and efficiency of the assay. The possibility of implementing the assay on a portable, highly sensitive AC susceptometer platform was also investigated. The performance of the assay under these circumstances was compared with that using a superconducting quantum interference device (SQUID); the sensitivity of the assay was similar for both platforms. It is concluded that, the VAM-NDA opens up the possibility to perform biomolecular detection in point-of-care and outpatient settings on portable platforms similar to the one tested in this thesis. Finally, the VAM-NDA was used to detect Escherichia coli bacteria and the spores of Bacillus globigii, the non-pathogenic simulant of Bacillus anthracis. A limit of detection of at least 50 bacteria or spores was achieved. This shows that the assay has great potential for sensitive detection of biomolecules in both environmental and biomedical applications.

A method for extraction of proteins from dried blood spots (DBS) for analysis using Proseek is developed and evaluated. DBS, as sample format, possesses a number of desirable advantages over for example plasma samples. These advantages include for example minimal patient invasiveness, sampling simplicity and non regulated sample transportation. Highly reproducible quantitative detection of 92 proteins is demonstrated from a 1.2 mm in diameter DBS disk. The DBS inter spot analysis precision (7% coefficient of variance) is comparable to plasma inter assay precision (6% coefficient of variance). Coefficient of variance is the ratio between standard deviation to mean value for the analysed replicates. Proseek analysis of DBS could possibly reveal a unique opportunity to examine health related issues in extremely premature infants hopefully resulting in increased survival rates in the future.

Here we present a novel Smartphone-based colorimeter and demonstrate its application to the measurements of glucose and protein concentrations in biological samples. The key innovation of our approach was to combine powerful image processing encoded into a mobile phone application with a low cost 3D printed sample holder that allowed to control lighting conditions and significantly improved sensitivity. Different solutions with protein and glucose concentrations ranging from 0 to 2000 mg/dL were prepared and analyzed using our system. The Smartphone-based colorimeter always correctly classified the corresponding reagent strip pads, what confirms that it can be used as a low cost alternative for commercial test strip analyzers.

Biomarkers are biological measures used for clinical assessment, whether an individual has a particular medical condition or to monitor and predict health states in individuals. Sensitive detection and quantification of various biomarkers are essential for disease diagnostics. The majority of biomarker-based diagnostics examines the presence and quantity of a single biomarker. Since the symptoms of many diseases are alike, multiplexed biomarker tests are highly desirable. Furthermore, detection of multiple biomarkers would improve the accuracy of diagnosis as well as providing additional information about the prognosis. Microarray platforms have the potential for higher level of multiplexing for biomarker detection. However, conventional microarray technologies are limited by the sensitivity of assays. This dissertation describes how single-particle interferometric reflectance imaging sensor (SP-IRIS) overcomes the sensitivity issues in biomarker detection and its applications to biomolecular and cellular biomarker detection assays. SP-IRIS provides optical detection of individual nanoparticles when they are captured onto a simple reflecting substrate, providing single-molecule sensitivity. This technique can be used to detect natural nanoparticles (such as viruses) without labels as well as molecular analytes (proteins and nucleic acids) that are labeled with metallic nanoparticles. Moreover, the advancements in technology make SP-IRIS ideal for the detection of low abundance biomarkers. Utilization of light polarization in combination with plasmonic gold nanorods as labels enhances the signal-to-noise ratio in nanoparticle detection allowing for the use of low numerical aperture optics increasing the field-of-view, hence, the throughput and sensitivity. Additionally, the integration of a disposable microfluidic flow cell and dynamic particle tracking in kinetic measurements provide a robust, ultra-sensitive and automated diagnostic platform. This dissertation focuses on the development of biological assays demonstrating effective use of SP-IRIS as a clinical diagnostic platform. We discuss the development of protein, nucleic acid and biological nanoparticle detecting SP-IRIS microarrays. We demonstrate four digital detection platforms for Hepatitis B, microRNA, rare mutations in an oncogene, KRAS, and virus-like particle detection with ultra-high sensitivity. / 2022-09-28T00:00:00Z