Discovery of a Highly Specific Anti-methotrexate (MTX) DNA Aptamer for Antibody-Independent MTX Detection.

High-dose methotrexate (MTX) therapy is used to treat a wide variety of cancers such as leukemia and lymphoma, while the resulting high blood concentration of MTX faces a risk of life-threatening side effects, so it is essential to monitor the concentration carefully. Currently, the MTX concentration is measured using antibody-based kits in a clinical setting; however, the heterogeneity and batch-to-batch variation of antibodies potentially compromise the detection limit. Here, we developed MTX detection systems with chemically synthesizable homogeneous oligonucleotides. Microbead-assisted capillary electrophoresis (MACE)-SELEX against MTX successfully identified MSmt7 with a similar level of specificity to anti-MTX antibodies within three rounds. The 3'-end of MSmt7 was coupled to a peroxidase-like hemin-DNAzyme to construct a bifunctional oligonucleotide for MTX sensing, where MTX in 50% human serum was detected with a limit of detection (LoD) of 118 nM. Furthermore, amplifying the DNAzyme region with rolling circle amplification significantly improved the sensitivity with an LoD of 290 pM. Presented oligonucleotide-based MTX detection systems will pave the way for antibody-independent MTX detection with reliability and less cost in the laboratory and the clinic.

Wafer-scalable chemical modification of amino groups on graphene biosensors.

Graphene's remarkable attributes make it suitable for application to biosensors for biomolecular recognition. Specific and precise target detection is realized by designing robust methods for immobilization of probe molecules, such as oligonucleotides, antibodies, receptors, and sugar chains, to a device surface. In this research, we developed a chemical modification method with a plasma treatment of amino groups on natural defects of graphene, which is compatible with a wafer-scalable semiconductor process, to prevent deterioration of the carrier mobility. The plasma treatment was optimized in terms of the efficiency of the amino radical generation, length of the mean free path, and reaction energy on graphene. The density of the modified amino groups on graphene was approximately 0.065 groups/nm2, and the change in the ΔId/ΔVg characteristic of the graphene field-effect transistor (FET) was negligible. DNA probes were then attached to the amino groups on the graphene FET. The target complementary DNA was detected at 1 nM after hybridization using the graphene FET devices. The plasma-assisted modification of the amino groups on the graphene surface was developed for immobilization of the DNA probes, and hybridization with the target DNA was demonstrated without deterioration of the carrier mobility.

Ionic Liquids with Wafer-Scalable Graphene Sensors for Biological Detection.

Ionic liquids, known as non-volatile solvents, have potential for realizing microanalysis with a minute quantity of sample. Here we report the measurement of the Id-Vg characteristics during the enzymatic catalytic reactions and streptavidin-biotin binding in ionic liquids by using the graphene FET sensors we fabricated, and successfully monitored the biological reactions in much smaller amount of solvents. These findings suggest the possibility of ionic liquids for application in bio-microanalysis with high sensitivity.

Bromocriptine-responsive Cushing's disease; clinical and biochemical remission accompanied by amelioration of impaired ocular movement.

A 73-year-old woman presented with disturbance of right eyelid opening with ptosis and impairment of right ocular movement, and later was found to have Cushingoid features. Endocrine examinations revealed that plasma ACTH and cortisol levels were elevated, lack of circadian rhythm, resistant to low-doses (0.5, 1, 2 mg) and high-dose (8 mg) dexamethasone, and responsive to CRH and DDAVP. Magnetic resonance imaging (MRI) revealed a large pituitary tumor invading the right cavernous sinus. After two months treated with bromocriptine (5 mg/day), she showed clinical improvement with normalization of plasma ACTH and cortisol levels, and improvement of right eyelid opening and ocular movement. MRI, however, revealed no apparent reduction in the size of pituitary tumor. This is a rare case of bromocriptine-responsive Cushing's disease.