Label-Free Detection of Biomarkers in Human Serum and a New Drug Delivery System Based on Reverse Electrodialysis

Abstract

Part 1: Label-Free Detection of Biomarkers in Human Serum A rapid and sensitive detection as well as quantification of peptides and proteins from complex biological samples is of importance for disease diagnosis. Such disease-associated macromolecules, existing in very low levels in biological samples, relate their concentration with the degree of disease progression. To monitor the level of peptides and proteins, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used owing to its high sensitivity (< fmol) and time-saving properties with a wide detection range of molecular mass. However, MALDI-TOF MS suffers from interferences of a variety of highly abundant peptides, proteins, salts, and other contaminants when detecting target molecules with extremely low concentration (~nM) directly from complex biosamples. As such, preseparation and enrichment of proteins are essential for the detection of these low abundant proteins prior to MS analysis. Label-free sensing technologies provide rapid detection of multiple biomarkers in complex biosamples such as cerebrospinal fluid, urine, and blood. Nonspecific bindings are an important issue in the case of the label-free detection methodologies because any species adsorbed on the sensor surface may produce a signal, leading to the reduced accuracy and reliability of sensors. Thus, identification of the degree of disease progression and diagnosis become difficult. In this study, we functionalized Au-plated magnetic microspheres (MGMs) with self-assembled monolayers (SAMs) for the effective prevention of the nonspecific bindings in human serum. The SAM employed in this study was composed of a chain of oligo ethylene glycol for preventing non-specific bindings and terminal carboxyl group for antibody immobilization. The antibody-conjugated MGMs were exposed to human serum to enrich target antigens specifically and the antigen-conjugated MGMs were directly analyzed by MALDI-TOF MS. To investigate the degree of nonspecific bindings and target enrichment, we compared the MGMs with four types of commercial magnetic microbeads. As the results, the MGMs showed superior enrichment properties out of the beads tested while providing effective suppression of the nonspecific bindings. Especially, the detection limit of myoglobin was 0.5 ng/mL in human serum using the MGMs. Finally, we conducted multiple detection of biomarkers in human serum simultaneously by taking both advantages of MALDI-TOF MS (label-free detection) and the MGMs (suppression of nonspecific bindings).

Part 2: A New Drug Delivery System Based on Reverse Electrodialysis Recently, reverse electrodialysis (RED) has been attracted great attention of scientists as an alternative way of power production due to use of inexhaustible resources (seawater and river water) and stable power production. However, there are still obstacles in scale-up applications, for example, high cost and short lifetime of ion-exchange membranes and low power density compared with the current power production methods. In this study, we developed a new transdermal drug delivery system based on RED as an electrical power source. For the purpose of power production in a large scale, RED system requires electrodes at the ends of the cell to relay the generated power to an external circuit. However, a miniaturized RED patch for transdermal drug delivery does not involve any metal electrodes to introduce the electrical power into a biological system because both systems comprise only ionic current. Thus, the patch-type RED system that generates a low power can be suitably utilized for the purpose of transdermal drug delivery. The penetration rate of risedronate (RIS) was 36-fold higher with the RED patch than with a diffusive patch. We finally conducted in vivo experiments using osteoporosis-induced female mice by removing the ovary. As the results, the mice treated with RIS by means of the RED patch were effectively prevented from proceeding osteoporosis while the mice treated with RIS only by passive diffusion led to very similar results to the ovary-removed mice, showing almost no therapeutic effects.