In this study, a field effect transistor (FET)-type biosensor based on 0.5 mm standard complementary metal oxide semiconductor (CMOS) technology is proposed and its feasibility for detecting deoxyribonucleic acid (DNA) and protein molecules is investigated. Au, which has a chemical affinity with thiol by forming a self-assembled monolayer (SAM), was used as the gate metal in order to immobilize DNA and protein molecules. A Pt pseudo-reference electrode was employed for the detection of biomolecules. The sensor was fabricated as a p-channel (P)MOSFETtype because PMOSFET with positive surface potential is useful for detecting negatively charged biomolecules from the view point of its high sensitivity and fast response time. DNA and protein molecules were detected by measuring the variation of the drain current due to the variation of biomolecular charge and capacitance. DNA and protein molecules used in the experiment were 15mer -oligonucleotide probe and streptavidin-biotin protein complexes, respectively. DNA was detected by both in situ and ex situ measurements. Additionally, to verify the interactions among SAM, streptavidin, and biotin, surface plasmon resonance (SPR) measurement was performed.
We have fabricated field effect transistor (FET)-type biomolecular sensor for the detection of the deoxyribonucleic acid (DNA) sequence based on 0.5 µm standard complementary metal oxide semiconductor (CMOS) technology and investigated its electrical characteristics. A Pt reference electrode with improved performance was employed for the detection of the DNA sequence and Au, which has a chemical affinity with thiol by forming a self-assembled monolayer (SAM), was used as the gate metal in order to immobilize the DNA. It was fabricated as a p-channel metal oxide semiconductor (PMOS) FET-type because PMOSFET with positive surface potential could be very attractive for detecting negatively charged DNA from the view point of high sensitivity and fast response time. The FET-based biomolecular sensor can detect the DNA sequence by measuring the variation of drain current due to a biomolecular charge after DNA probe immobilization and variation of capacitance after DNA hybridization. The gate potential of the sensor was applied by the Pt reference electrode and DNA was detected by both in situ and ex situ measurements. The drain current increased when a single-stranded DNA (ss-DNA) with thiol was immobilized because the effect of DNA charge with thiol is dominant. The drain current decreased when the DNA was hybridized into a double-stranded DNA (ds-DNA) because of the decrease in capacitance due to DNA hybridization. In situ measurement showed good agreement with ex situ measurement.
To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.
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