Biomedical subjects

Masahiko Hashimoto

Publications and source records attributed to Masahiko Hashimoto.

13 recordsLinked to original sources

PubMed2006

On-demand patterning of protein matrixes inside a microfluidic device.

On-demand immobilization of proteins at specific locations in a microfluidic device would advance many types of bioassays. We describe a strategy to create a patterned surface within a microfluidic channel by electrochemical means, which enables site-specific immobilization of protein matrixes and cells under physiological conditions, even after the device is fully assembled. By locally generating hypobromous acid at a microelectrode in the microchannel, the heparin-coated channel surface rapidly switches from antibiofouling to protein-adhering. Since this transformation allows compartmentalizing of multiple types of antibodies into distinct regions throughout the single microchannel, simultaneous assay of two kinds of complementary proteins was possible. This patterning procedure can be applied to conventional microfluidic devices since it requires only some electrodes and a voltage source (1.7 V, DC).

Animals↗

PubMed2006

Merging microfluidics with microarray-based bioassays.

Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.

Biological Assay↗

PubMed2006

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations.

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.

Animals↗

PubMed2005

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations.

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K-ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time=19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.

Biosensing Techniques↗

PubMed2005

Patterning the surface cytophobicity of an albumin-physisorbed substrate by electrochemical means.

Spatiotemporal control of surface properties under physiological conditions such as those found in culture media is an important technique in fundamental cell biology, tissue engineering, and cell-based bioelectronics. To this end, we have developed a mild, wet cellular micropatterning technique. The principle of the technique is based on the fact that the cell-repellant property of the albumin-coated substrate rapidly switches to cell-adhesive upon exposure to the reactive oxidizing agent, electrochemically generated hypobromous acid. Herein, we report the effect of the hypobromous acid on serum albumin physisorbed on a hydrophobic substrate. It was found that albumin molecules detach from the substrate by application of the oxidizing agent, resulting in exposure of the underlying hydrophobic surface to the liquid phase. The adsorption of extracellular matrix proteins such as fibronectin onto the hydrophobic surface induces cell adhesion and growth.

Adsorption↗

PubMed2005

Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations.

We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.

Base Sequence↗

PubMed2005

Induction of protective immunity to anthrax lethal toxin with a nonhuman primate adenovirus-based vaccine in the presence of preexisting anti-human adenovirus immunity.

Prevention or therapy for bioterrorism-associated anthrax infections requires rapidly acting effective vaccines. We recently demonstrated (Y. Tan, N. R. Hackett, J. L. Boyer, and R. G. Crystal, Hum. Gene Ther. 14:1673-1682, 2003) that a single administration of a recombinant serotype 5 adenovirus (Ad) vector expressing anthrax protective antigen (PA) provides rapid protection against anthrax lethal toxin challenge. However, approximately 35 to 50% of humans have preexisting neutralizing antibodies against Ad5. This study assesses the hypothesis that a recombinant adenovirus vaccine based on the nonhuman primate-derived serotype AdC7, against which humans do not have immunity, expressing PA (AdC7PA) will protect against anthrax lethal toxin even in the presence of preexisting anti-Ad5 immunity. Naive and Ad5-immunized BALB/c mice received (intramuscularly) 10(8) to 10(11) particle units (PU) of AdC7PA, Ad5PA (a human serotype Ad5-based vector expressing a secreted form of PA), or AdNull (an Ad5 vector with no transgene). Robust anti-PA immunoglobulin G and neutralizing antibodies were detected by 2 to 4 weeks following administration of AdC7PA to naive or Ad5 preimmunized mice, whereas low anti-PA titers were detected in Ad5-preimmunized mice following administration of Ad5PA. To assess protection in vivo, naive or mice previously immunized against Ad5 were immunized with AdC7PA or Ad5PA and then challenged with a lethal intravenous dose of Bacillus anthracis lethal toxin. Whereas Ad5PA protected naive mice against challenge with B. anthracis lethal toxin, Ad5PA was ineffective in mice that were previously immunized against Ad5. In contrast, AdC7PA functioned effectively not only to protect naive mice but also to protect Ad5-preimmunized mice, with 100% survival after lethal toxin challenge. These data suggest the nonhuman-based vector AdC7PA is an effective vaccine for the development of protective immunity against B. anthracis and importantly functions as a "sero-switch" base for an adenovirus vaccine to function in the context of preexisting anti-Ad immunity.

Adenoviridae↗

PubMed2004

Rapid PCR in a continuous flow device.

Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 degrees C) and renaturation (55 degrees C-68 degrees C) zones; above 6 mm s(-1) the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from lambda-DNA. Amplifications at velocities ranging from 1 mm s(-1) to 20 mm s(-1) were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle(-1)) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle(-1)). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).

Bacteriophage lambda↗

PubMed2003

Second-line chemotherapy with low-dose CPT-11 and cisplatin for colorectal cancer resistant to 5-FU-based chemotherapy.

PURPOSE: With the aim of reducing the toxicities of irinotecan (CPT-11) while maintaining its antitumor effect, we treated colorectal cancer patients resistant to chemotherapy based on 5-fluorouracil (5-FU) with low-dose CPT-11 and cisplatin (CDDP). METHODS: CPT-11 (27 mg/m2) and CDDP (6 mg/m2) were administered on days 1, 8 and 15 every 4 weeks to 20 patients with recurrent or metastatic colorectal cancer. When toxicities were noted, administrations were delayed or the dose was reduced. RESULTS: No severe toxicity (i.e. grade 3 or more) was observed in this study. Nausea was observed in 50% of patients (10/20) and fatigue in 30% (6/20). Only four patients developed leukopenia (three grade 1 and one grade 2). Although the overall response rate was 15% (three partial response, seven no change, and ten progressive disease), the median time to progression was 7.0 months and the median survival time was 18.0 months. The treatment was well tolerated as outpatient therapy. CONCLUSION: Low-dose CPT-11 and CDDP treatment should be considered as second-line chemotherapy for patients with recurrent or metastatic colorectal cancer resistant to 5-FU-based chemotherapy.

Adult↗

PubMed2003

On-line integration of PCR and cycle sequencing in capillaries: from human genomic DNA directly to called bases.

A fully integrated system has been developed for genetic analysis based on direct sequencing of polymerase chain reaction (PCR) products. The instrument is based on a serially connected fused-silica capillary assembly. The technique involves the use of microreactors for small-volume PCR and for dye-terminator cycle-sequencing reaction, purification of the sequencing fragments, and separation of the purified DNA ladder. Four modifications to the normal PCR protocol allow the elimination of post-reaction purification. The use of capillaries as reaction vessels significantly reduced the required reaction time. True reduction in reagent cost is achieved by a novel sample preparation procedure where nanoliter volumes of templates and sequencing reaction reagent are mixed using a micro- syringe pump. The remaining stock solution of sequencing reaction reagent can be reused without contamination. The performance of the whole system is demonstrated by one-step sequencing of a specific 257-bp region in human chromosome DNA. Base calling for the smaller fragments is limited only by the resolving power of the gel. The system is simple, reliable and fast. The entire process from PCR to DNA separation is completed in approximately 4 h. Feasibilities for development of a fully automated sequencing system in the high-throughput format and future adaptation of this concept to a microchip are discussed.

DNA↗

PubMed2003

[Effect of cimetidine with chemotherapy on stage IV colorectal cancer].

We herein report the result of a prospective study to investigate the efficacy of cimetidine administration in conjunction with chemotherapy for stage IV colorectal cancer. Sixty-two patients treated with Leucovorin/5-fluorouracil therapy were enrolled from 1996 to 2000. Both groups were well matched for pre-treatment characteristics. There was no difference in survival in cur B patients. However, the cimetidine group had significantly prolonged survival in the patients with cur C or non-resectable carcinoma. This study suggests that cimetidine treatment may improve the survival of patients with non-curative surgery for stage IV colorectal cancer.

Adjuvants, Immunologic↗

PubMedDate not supplied

Fas ligand expression is a risk factor for liver metastasis in colorectal cancer with venous invasion.

The expression of Fas ligand (Fas L) was investigated in sixty-seven patients with colorectal cancer for possible correlation with liver metastasis. Fas L expression was positive in 48 and negative in 19 cases. In Fas L-positive cases, 14 patients had liver metastasis including metachronous metastasis. However, only one case had liver metastasis in Fas L-negative patients (p = 0.0344). In colorectal cancer with venous invasion, 13 from 25 Fas L-positive cases had liver metastasis but only one in 19 negative cases (p = 0.0003). In the cases without venous invasion, no liver metastasis was observed in Fas L-negative cases. These results suggest that Fas L expression may be a predictive factor for liver metastasis in colorectal cancer with venous invasion.

Adult↗

PubMedDate not supplied

Prophylactic efficacy of allopurinol ice ball for leucovorin/5-fluorouracil therapy-induced stomatitis.

BACKGROUND: Stomatitis in cancer chemotherapy manifests with pronounced subjective symptoms, lowers the patient's quality of life (QOL) and may necessitate the discontinuation of chemotherapy. There have been few effective therapies established to date for chemotherapy-induced stomatitis. PATIENTS AND METHODS: We used frozen allopurinol solution (allopurinol ice balls) in the prevention of stomatitis associated with leucovorin (LV)/5-fluorouracil (5-FU) therapy for colon cancer. RESULTS: Among 32 patients undergoing LV/5-FU therapy, without the use of the allopurinol ice balls, 15 patients developed stomatitis. On the other hand, only 3 patients developed stomatitis among 20 patients receiving the chemotherapy in conjunction with allopurinol ice balls (p=0.0187). Seven patients who developed stomatitis during a course of chemotherapy without allopurinol ice balls were administered the ice balls in the subsequent course; 6 of these patients responded with lessened severity of stomatitis. In 2 of these responders, stomatitis of comparable severity recurred on discontinuation of the medication. CONCLUSION: Concomitant use of allopurinol ice balls is effective in preventing stomatitis in patients undergoing LV/5-FU therapy.

Allopurinol↗