Modification of the glass surface property in PDMS-glass hybrid microfluidic devices.

This paper presents a simple method to change the hydrophilic nature of the glass surface in a poly(dimethylsiloxane) (PDMS)-glass hybrid microfluidic device to hydrophobic by an extra-heating step during the fabrication process. Glass substrates bonded to a native or oxygen plasma-treated PDMS chip having microchambers (12.5 mm diameter, 110 µm height) were heated at 200°C for 3 h, and then the hydrophobicity of the glass surfaces on the substrate was evaluated by measuring the contact angle of water. By the extra-heating process, the glass surfaces became hydrophobic, and its contact angle was around 109°, which is nearly the same as native PDMS surfaces. To demonstrate the usefulness of this surface modification method, a PDMS-glass hybrid microfluidic device equipped with microcapillary vent structures for pneumatic manipulation of droplets was fabricated. The feasibility of the microcapillary vent structures on the device with the hydrophobic glass surfaces are confirmed in practical use through leakage tests of the vent structures and liquid handling for the electrophoretic separation of DNA molecules.

Application of cell-free expression of GFP for evaluation of microsystems.

Coupled cell-free transcription-translation (CFTT) of green fluorescent protein (GFP) has been applied as a reporter system to microfluidic chip-related technologies. In polymerase chain reaction (PCR)-based biomolecular logic gate system, in which addition of primer set and amplification of PCR product represent input and output signal respectively, GFP gene was inserted in the template DNA, which was then amplified, transcribed and translated to GFP. The green fluorescence reported as if the amplification has occurred or not, that is, the fluorescence reports positive output signal. CFTT of GFP was also adopted to evaluate on-chip capillary electrophoresis (CE)-based DNA fractionation, which was developed to isolate single DNA species from reaction mixture of DNA ligase-catalyzed DNA-assembly. As a model system, GFP gene was inserted in the target DNA fragment. The collected fraction was amplified with PCR and subjected to a CFTT system, and green fluorescence was observed showing that the fractionation was successful. These results showed that CFTT of GFP is a useful tool to verify, estimate, and monitor microfluidic chip-related technologies in which cell-free protein synthesis is involved.

A rapid method for optimizing running temperature of electrophoresis through repetitive on-chip CE operations.

In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method.

Pneumatic handling of droplets on-demand on a microfluidic device for seamless processing of reaction and electrophoretic separation.

Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.

Millisecond denaturation dynamics of fluorescent proteins revealed by femtoliter container on micro-thermodevice.

Real-time observation of biomolecular behavior focusing on high speed temperature response is an essential endeavor for further biological study at the molecular level. This is because most of the important biological functions at the molecular level happen at the sub-second time scale. We used our own on-chip microheaters and microcontainers to observe the denaturation dynamics of fluorescent proteins at the millisecond time scale. The microheater controls the temperature in 1 ms under the microscope. Fluorescent proteins were contained in 28 fL PDMS microcontainers to prevent them from diffusing into the solution. The proteins were denatured by high temperatures and observed by a high speed CCD camera with 5 ms per frame. Hence, denaturation speeds of red fluorescent proteins (rDsRed and rHcRed) were measured to be 5-10 ms. Green fluorescent proteins (rAcGFP and rGFPuv) denatured with bi-exponential decay. rAcGFP denatured with time constants of 5 ms and 75 ms while rGFPuv denatured with 10 ms and 130 ms. This may be the reverse process of a two step renaturation of GFP observed in a previous report. This micro-thermodevice is applicable to other biomaterials such as nucleic acids or other proteins. It does not require any chemical treatment nor mutation to the biomaterial itself. Therefore, the methodology using this general purpose device gives access to biomolecular studies in short time scales and acts as a powerful tool in molecular biology.

Polymerase chain reaction-based biochemical logic gate coupled with cell-free transcription-translation of green fluorescent protein as a report gate.

Polymerase chain reaction-based biochemical logic gates were designed for AND, OR, NOT, and AND-NOT operations, whose output signal is reported by coupled cell-free transcription-translation of green fluorescent protein.

Evaluation of cell-free protein synthesis using PDMS-based microreactor arrays.

A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.

Application of on-chip capillary electrophoresis to cell-free preparation of recombinant DNA.

An on-chip capillary electrophoresis-based DNA collection was applied to the isolation of target DNA species from a DNA mixture generated by a polymerase chain reaction (PCR), whose starting material was a ligation mixture of an insert and an expression vector. The collected DNA was then amplified by PCR and properly worked as template DNA in a coupled cell-free transcription/translation system. These results demonstrateed that total operation in standard genetic engineering can be performed in a cell-free condition.

Reactivity of ferrocenylcarbodiimide with DNA duplex containing single-mismatched base pairs.

Ferrocenylcarbodiimide (1), which is known to react with a guanine (G) or thymine (T) base of single stranded DNA, was allowed to react with DNA duplex having a single mismatched base pair of G-T, T-T, or T-cytosine (C). Electrophoreograms of the reaction mixture showed that 1 could react with G or T base of the mismatched sites on the DNA duplex. However, 1 also reacted with the G base of the terminal site on the DNA duplex. This showed that 1 can react with an unpaired base or unstable base pair such as a terminal or mismatched base on the DNA duplex. Electrochemical mismatch detection could be achieved after hybridization of the ferrocenylated mismatched DNA duplex with a selected DNA probe-immobilized electrode. These results revealed that 1 has a potentiality of serving as a labeling reagent of mismatched bases on the DNA duplex, which is important in the search for heterozygous single nucleotide polymorphisms (SNPs).

Detection of cRNA hybridized on a DNA chip using a tetrakis-acridinyl peptide cassette, consisting of TAP and partially self-complementary oligonucleotide, d[A18(TA)51].

A tetrakis-acridinyl peptide (TAP) cassette, consisting of a double-stranded region of alternating AT sequence bound to TAP and a single stranded overhanging sequence of continuous dA, was prepared by mixing TAP with d[A18(TA)51]. A TAP cassette could be applied to the fluorometric detection of hybridized DNA on the DNA chip, which was prepared by stamping a 45-meric DNA probe onto a gold-coated plastic chip using a high-precision spotter developed at RIKEN. Spots on the DNA chip were imaged by a CCD camera after hybridization with 65-meric target single-stranded DNAs carrying a continuous dA20 sequence (dA tail) on the DNA chip after treatment with a TAP cassette. Their fluorescence intensity on the DNA chip showed a good linear correlation with the concentration of the target DNAs in the range from 10 pM to 1 nM. Fluorescence of their spots derived from the TAP cassette remaining on the surface of the DNA chip through the dA tail of the hybridized target DNA. Furthermore, the TAP cassette could be successfully applied to the quantitative detection of complementary RNAs (cRNAs) prepared from rat brain with reverse transcription and in vitro transcription.

Fluorescence energy transfer probes based on the guanine quadruplex formation for the fluorometric detection of potassium ion.

Dual-labeled oligonucleotide derivative, FAT-0, carrying 6-carboxyfluorescein (FAM) and 6-carboxy-tetramethylrhodamine (TAMRA) labels at 5'- and 3'-termini of thrombin-binding aptamer (TBA) sequence 5'-GGTTGGTGTGGTTGG-3' and its derivatives, FAT-n (n=3, 5, and 7) were designed and synthesized. FAT-n derivatives contained a T(m)A spacer (m=2, 4, and 6, respectively) at 5'-end of TBA sequence. The probes were developed to estimate the spacer effect on FRET efficiency and to identify the best probe for sensing of K(+). Circular dichroism (CD), UV-vis absorption, and fluorescence studies revealed that all FAT-n probes could form the intramolecular tetraplex structures after binding K(+). Association constants of particular K(+)/FAT-n complexes were determined using different experimental approaches. Suitability of particular probes for sensitive monitoring of K(+) in intra- and extracellular conditions was examined and discussed. Calibration graphs of fluorescence ratio were linear in the K(+) concentration range of 2-10 mM for extracellular conditions showing sensitivity of 1.2% mM(-1) K(+) and for intracellular conditions in the range of 100-200 mM with sensitivity of 0.49% mM(-1) K(+).

Direct modification of mRNA by ferrocenyl carbodiimide and its application to electrochemical detection of mRNA.

Ferrocenyl carbodiimide (1) could be used for the direct labeling of synthetic RNA and expressed mRNA in vitro with the electrochemically active ferrocene moieties. These RNAs modified by 1 could be detected electrochemically coupled with a DNA probe-immobilized electrode. After hybridization of 1.1 Kb mRNA modified by 1 with the DNA probe-immobilized electrode, the peak charge observed by an Osteryoung square wave voltammetry (SWV) measurement correlated well with the concentration of mRNA, having a detection limit at the sub nanogram level.

Electrochemical detection of DNase I activity.

DNase I in one microl of the water could quantitate electrochemically with the detection limit of 0.01 units (ca. 20 pg) by using the ferrocenyl oligonucleotide-immobilized electrode prepared by thiolated oligonucleotide and ferrocenyl carbodiimide as a simple labeling reagent of redox unit.

Electrochemical SNP detection.

Detection of mismatched base on a DNA duplex, which is important for the search of the heterozygote, achieved by the comparison with the reactivity of ferrocenylcarbodiimide (1) developed by our group. Mismatched thymine base on the 30-meric DNA duplex as a model of SNPs in lipoprotein lipase (LPL) gene, could react with 1 in 20 mM borate buffer (pH 8.5) and 0.1 M NaCl containing 20% DMSO at 37 degrees C. DNA duplex modified by 1 in the mismatched thymine bases could be detected by its shifted migration time in microTAS electrophoresis.

Controlling the expression ratio of two proteins by inserting a terminator between the two genes.

An approach was developed in controlling the expression ratio of two genes encoded in DNA by inserting a terminator sequence between the two genes. Escherichia coli ribosomal RNA operon T1 terminator, or E. coli tryptophane attenuater was introduced between two green fluorescent protein (GFP) mutants: ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation reflected the termination efficiency of the transcriptional termination activity of the inserted terminator.

G quadruplex-based FRET probes with the thrombin-binding aptamer (TBA) sequence designed for the efficient fluorometric detection of the potassium ion.

The dual-labeled oligonucleotide derivative, FAT-0, carrying 6- carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at the 5' and 3' termini of the thrombin-binding aptamer (TBA) sequence 5'-GGT TGG TGT GGT TGG-3', and its derivatives, FAT-n (n=3, 5, and 7) with a spacer at the 5'-end of a TBA sequence of T(m)A (m=2, 4, and 6) have been designed and synthesized. These fluorescent probes were developed for monitoring K(+) concentrations in living organisms. Circular dichroism, UV-visible absorption, and fluorescence studies revealed that all FAT-n probes could form intramolecular tetraplex structures after binding K(+). Fluorescence resonance energy transfer and quenching results are discussed taking into account dye-dye contact interactions. The relationship between the fluorescence behavior of the probes and the spacer length in FAT-n was studied in detail and is discussed.

Preparation of carbodiimide-terminated dithiolane self-assembly monolayers as a new DNA-immobilization method.

A carbodiimide derivative having a dithiolane part at its terminus was designed and synthesized for use to construct carbodiimide-coated self-assembly monolayers (SAMs) on a gold surface with 6-mercaptohexanol (6MH). When treated with poly(dT), poly(dA), or poly(dA)poly(dT), only poly(dT) was immobilized on the surface of the SAMs through a specific reaction of the free imino moiety of thymine (T) with the carbodiimide moiety. The carbodiimide-covered SAM treated with probe DNA was tested in hybridization with sample DNA. Its hybridization efficiency was estimated by ferrocenylnaphthalene diimide (FND), described previously and the result revealed that the carbodiimide-covered SAM electrode can immobilize a DNA probe through the thymine moiety not involved in base pairing. The resulting electrode was capable of hybridizing with the target DNA, as proven by an increased current response of FND.

Immobilization of RNase S-Peptide on a single-stranded DNA-fixed gold surface and effective masking of its surface by an acridinyl poly(ethylene glycol).

Oligonucleotide-peptide conjugate was synthesized by coupling of RNase S-peptide to a 24-mer single-stranded DNA (ssDNA) oligonucleotide to be immobilized on its complementary ssDNA oligonucleotide-fixed gold surface of sensor chip or electrode. Immobilization of on the ssDNA-fixed gold surface through DNA duplex formation was confirmed by quartz crystal microbalance (QCM) and electrochemical measurements. After treating with a synthetic acridinyl poly(ethylene glycol) (APEG), specific interaction of S-protein with the S-peptide immobilized on the gold surface was demonstrated by QCM without nonspecific adsorption of unrelated proteins such as BSA and RNase A at the surfaces. This result suggested that the acridine parts of APEG could bind to the DNA duplex on the gold surface and the poly(ethylene glycol) parts were fastened on the surface to resist the adsorption of proteins. Thus, the combination of oligonucleotide-peptide conjugate, ssDNA-fixed chip and APEG with effective masking property provides a new tool for the analysis of specific peptide-protein interactions without disturbance by other unrelated proteins.

Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene.

Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16(Ink4a)) were used to be immobilized on the electrochemical array (ECA) chip. Using 1 microL of 10 ng/microL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.

Tetrakis-acridinyl peptide: distance dependence of photoinduced electron transfer in deoxyribonucleic acid assemblies.

The distance dependence of photoinduced electron transfer in deoxyribonucleic acid (DNA) duplex was investigated using the "TAP cassette" systems of the general formula (AT)6A(n)XA(9-n) (X denote guanine (G) or cytosine (C)). The tetrakis-9-acridinyl peptide (TAP) binds tightly with (AT)6 duplex region showing strong fluorescence that was not quenched by the A(n)XA(9-n) single-stranded region. Quenching was observed after duplex formation with the complementary T(9-n)XT(n) strand (G-C pairing), showing clear dependence on the distance between the TAP and a guanine. An extremely low beta value of 0.22 was obtained in our electron transfer (ET) system that suggests exceptional good mediation of ET process. Experiments with G-mismatches showed negligible quenching for systems with guanine separated by more than one AT base pair that indicated rather inefficient ET process for duplexes containing disrupted pi-electronic system.