Improvement of limit of detection in primer extension-based multiplexed mutation assay using capillary electrophoresis.

One of the challenges in liquid biopsy for early cancer detection is ascribed to the fact that mutation DNA often represents an extremely small ratio of less than 1% compared to wild-type genes in blood. However, in conventional fragment analysis with capillary electrophoresis (CE), the detectable allele frequency could be about 5%. In this work, we developed an original reagent-based fragment analysis with single base extension (SBE) reactions for cancer-associated mutation assay using a commercially available CE device, and investigated on a possibility of improvement of limit of detection (LOD) for genetic mutation. First, after adjustment of reagent conditions for the SBE reactions, the linear relationship between gene template concentration and fluorescence intensity was obtained from 1 to 100 fmol of target genes. Next, from the results of an experiment to detect mutation EGFR L858R at abundance ratios of mutant type to wild type (100-fmol template) of 0, 1, 5, and 10%, it was shown that the target gene can be detected with LOD of 0.33%. This high sensitivity was realized in part by separating fluorescently labeled substrates into an individual tube for an each-colored SBE reaction. Moreover, mutations EGFR L858R and KRAS G12V were simultaneously detected at sensitivities equivalent to LODs of 0.57 and 0.47%, respectively. These results indicate that < 1% of mutations in multiplex gene mutations can be simultaneously detected, and that possibility suggests that the developed method can be used in clinical practice for detecting cancers.

An ultra-small nine-color spectrometer with a two-layer biparted ten-dichroic-mirror array and an image sensor.

An ultra-small (54 × 58 × 8.5 mm) and large aperture (1 × 7 mm) nine-color spectrometer-using an array of ten dichroic mirrors "biparted" as two layers-was developed and used for snapshot spectral imaging. Incident-light flux with a cross section smaller than the aperture size is split into nine color fluxes with 20-nm-width contiguous wavelength bands and central wavelengths of 530, 550, 570, 590, 610, 630, 650, 670, and 690 nm. Images of the nine color fluxes are simultaneously and efficiently measured by an image sensor. Unlike a conventional dichroic-mirror array, the developed dichroic-mirror array has a unique biparted configuration that not only increases the number of colors that can be measured simultaneously but also improves the image resolution of each color flux. The developed nine-color spectrometer was used for four-capillary-array electrophoresis. Eight dyes concurrently migrating in each capillary were simultaneously quantified by nine-color laser-induced fluorescence detection. Since the nine-color spectrometer is not only ultra-small and inexpensive but also has high light throughput and sufficient spectral resolution for most spectral-imaging applications, it has the potential to be widely used in various fields.

Ultra-small four-emission-point spectral-detection system using seven-dichroic-mirror array.

An ultra-small and highly efficient spectral-detection system for four emission points was developed by integrating an injection-molded-plastic four-lens array, a seven-dichroic-mirror array, and an image sensor as one device. The seven-dichroic-mirror array was further miniaturized compared to our previous four-dichroic-mirror array by measures including reduction of the thickness of each dichroic mirror from 1.0 to 0.5 mm. As a result, the system enables highly sensitive and low-crosstalk seven-color detection of laser-induced fluorescence from four emission points of a four-capillary array. This capability allows simultaneous quantification of up-to-seven fluorophores concurrently present in each capillary. Sanger DNA sequencing and STR genotyping by four-capillary-array electrophoresis were experimentally demonstrated by the system.

Highly sensitive mutation quantification by high-dynamic-range capillary-array electrophoresis (HiDy CE).

A simple and robust ultra-small four-color-fluorescence detection system was developed by integrating its components, namely, a four-capillary array, an injection-molded-plastic four-lens array, a four-dichroic-mirror array, and a CMOS sensor, as one device. The developed system was applied to a high-dynamic-range capillary-array electrophoresis (HiDy CE) to quantify a rare EGFR mutant (MT) of exon 19 deletion in a large excess of EGFR wild type (WT). Samples with serially diluted MT and constant-concentration WT were co-amplified by competitive PCR and subjected to HiDy CE. The MT peak in each electropherogram was then compared to the WT peak. As a result, MT was quantified with high-sensitivity (LOD of 0.004% MT/WT) and four-orders-of-magnitude dynamic range (0.01-100% MT/WT) by HiDy CE. Moreover, compared with existing methods, HiDy CE achieves higher speed, higher sample throughput, and lower consumable cost per sample. It has therefore great potential to be used in clinical practice.

An ultra-small, multi-point, and multi-color photo-detection system with high sensitivity and high dynamic range.

Although multi-point, multi-color fluorescence-detection systems are widely used in various sciences, they would find wider applications if they are miniaturized. Accordingly, an ultra-small, four-emission-point and four-color fluorescence-detection system was developed. Its size (space between emission points and a detection plane) is 15 × 10 × 12 mm, which is three-orders-of-magnitude smaller than that of a conventional system. Fluorescence from four emission points with an interval of 1 mm on the same plane was respectively collimated by four lenses and split into four color fluxes by four dichroic mirrors. Then, a total of sixteen parallel color fluxes were directly input into an image sensor and simultaneously detected. The emission-point plane and the detection plane (the image-sensor surface) were parallel and separated by a distance of only 12 mm. The developed system was applied to four-capillary array electrophoresis and successfully achieved Sanger DNA sequencing. Moreover, compared with a conventional system, the developed system had equivalent high fluorescence-detection sensitivity (lower detection limit of 17 pM dROX) and 1.6-orders-of-magnitude higher dynamic range (4.3 orders of magnitude).

A simple and highly sensitive spectroscopic fluorescence-detection system for multi-channel plastic-microchip electrophoresis based on side-entry laser-beam zigzag irradiation.

A five-color fluorescence-detection system for eight-channel plastic-microchip electrophoresis was developed. In the eight channels (with effective electrophoretic lengths of 10 cm), single-stranded DNA fragments were separated (with single-base resolution up to 300 bases within 10 min), and seventeen-loci STR genotyping for forensic human identification was successfully demonstrated. In the system, a side-entry laser beam is passed through the eight channels (eight A channels), with alternately arrayed seven sacrificial channels (seven B channels), by a technique called "side-entry laser-beam zigzag irradiation." Laser-induced fluorescence from the eight A channels and Raman-scattered light from the seven B channels are then simultaneously, uniformly, and spectroscopically detected, in the direction perpendicular to the channel array plane, through a transmission grating and a CCD camera. The system is therefore simple and highly sensitive. Because the microchip is fabricated by plastic-injection molding, it is inexpensive and disposable and thus suitable for actual use in various fields.

Side-gated ultrathin-channel nanopore FET sensors.

A side-gated, ultrathin-channel nanopore FET (SGNAFET) is proposed for fast and label-free DNA sequencing. The concept of the SGNAFET comprises the detection of changes in the channel current during DNA translocation through a nanopore and identifying the four types of nucleotides as a result of these changes. To achieve this goal, both p- and n-type SGNAFETs with a channel thicknesses of 2 or 4 nm were fabricated, and the stable transistor operation of both SGNAFETs in air, water, and a KCl buffer solution were confirmed. In addition, synchronized current changes were observed between the ionic current through the nanopore and the SGNAFET's drain current during DNA translocation through the nanopore.

Side-entry laser-beam zigzag irradiation of multiple channels in a microchip for simultaneous and highly sensitive detection of fluorescent analytes.

A simple and highly sensitive technique for laser-induced fluorescence detection on multiple channels in a plastic microchip was developed, and its effectiveness was demonstrated by laser-beam ray-trace simulations and experiments. In the microchip, with refractive index nC, A channels and B channels are arrayed alternately and respectively filled with materials with refractive indexes nA for electrophoresis analysis and nB for laser-beam control. It was shown that a laser beam entering from the side of the channel array traveled straight and irradiated all A channels simultaneously and effectively because the refractive actions by the A and B channels were counterbalanced according to the condition nA < nC < nB. This technique is thus called "side-entry laser-beam zigzag irradiation". As a demonstration of the technique, when nC = 1.53, nA = 1.41, nB = 1.66, and the cross sections of both eight A channels and seven B channels were the same isosceles trapezoids with 97° base angle, laser-beam irradiation efficiency on the eight A channels by the simulations was 89% on average and coefficient of variation was 4.4%. These results are far superior to those achieved by other conventional methods such as laser-beam expansion and scanning. Furthermore, fluorescence intensity on the eight A channels determined by the experiments agreed well with that determined by the simulations. Therefore, highly sensitive and uniform fluorescence detection on eight A channels was achieved. It is also possible to fabricate the microchips at low cost by plastic-injection molding and to make a simple and compact detection system, thereby promoting actual use of the proposed side-entry laser-beam zigzag irradiation in various fields.

Slowing single-stranded DNA translocation through a solid-state nanopore by decreasing the nanopore diameter.

To slow the translocation of single-stranded DNA (ssDNA) through a solid-state nanopore, a nanopore was narrowed, and the effect of the narrowing on the DNA translocation speed was investigated. In order to accurately measure the speed, long (5.3 kb) ssDNA (namely, ss-poly(dA)) with uniform length (±0.4 kb) was synthesized. The diameters of nanopores fabricated by a transmission electron microscope were controlled by atomic-layer deposition. Reducing the nanopore diameter from 4.5 to 2.3 nm slowed down the translocation of ssDNA by more than 16 times (to 0.18 µs base(-1)) when 300 mV was applied across the nanopore. It is speculated that the interaction between the nanopore and the ssDNA dominates the translocation speed. Unexpectedly, the translocation speed of ssDNA through the 4.5 nm nanopore is more than two orders of magnitude higher than that of double-stranded DNA (dsDNA) through a nanopore of almost the same size. The cause of such a faster translocation of ssDNA can be explained by the weaker drag force inside the nanopore. Moreover, the measured translocation speeds of ssDNA and dsDNA agree well with those calculated by molecular-dynamics (MD) simulation. The MD simulation predicted that reducing the nanopore diameter to almost the same as that of ssDNA (i.e. 1.4 nm) decreases the translocation speed (to 1.4 µs base(-1)). Narrowing the nanopore is thus an effective approach for accomplishing nanopore DNA sequencing.

Dual-view imaging system using a wide-range dichroic mirror for simultaneous four-color single-molecule detection.

A dual-view imaging system for simultaneous four-color single-molecule (SM) detection was developed. As for the detection procedure, four species of SM fluorophores, namely, Alexa 488, 555, 647, and 680, are immobilized on different slides and excited by evanescent-wave illumination. Fluorescence emitted from an SM fluorophore is split by a wide-range dichroic mirror (WR DM) in a dual-view optics and imaged as two SM fluorescence spots (SM spots) on an electron-multiplying charge-coupled device (EM-CCD) at 100 Hz. The transmittance of the WR DM changes gradually over the wavelength range of 500 to 700 nm so that the signal ratios of the two SM spots for the four fluorophore species differ. A method for classifying SM fluorophores into four species in accordance with their signal ratios was developed. It was used to classify 597 SM fluorophores at an accuracy of above 98% for all the species. This accuracy is comparable to that of a conventional four-color SM detection system. To classify four species, the conventional system disperses SM fluorescence with a prism and provides an elongated SM spot that uses more pixels of an EM-CCD chip than that of the developed system. The developed system can thus detect 1.5-fold more SM spots with the same-size EM-CCD chip, so it can achieve 1.5-fold higher throughput. Moreover, the developed system is based on a simple and practical approach, namely, replacing an ordinary dichroic mirror in a commercially available dual-view optics with a WR DM. This replacement transforms a dual-view imaging system for two-color detection into a system for four-color detection. The developed system is suitable for detection systems of next-generation DNA sequencers and DNA microarray-chip analyzers.

Simultaneous four-color imaging of single molecule fluorophores using dichroic mirrors and four charge-coupled devices.

We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.

Improvement of biomolecule quantification precision and use of a single-element aspheric objective lens in fluorescence correlation spectroscopy.

We found a way to increase the precision with which biomolecules present at concentrations below 10(-10) M can be quantified by fluorescence correlation spectroscopy (FCS). The effectiveness of the way was demonstrated experimentally by using a single-element aspheric objective lens, which was newly developed to reduce the cost of FCS instruments. In the first part of this paper, the relative standard deviation (RSD) of FCS-based concentration measurements is estimated theoretically by an analytical approximation assuming the detection volume profiles in FCS setups to be Gaussian and by molecular simulations in which more realistic profiles are calculated from physical parameters of the measurement setups. In a limit of infinitely bright molecules and zero background emission, the analytical approximation predicts that the RSD at a concentration is minimized when the mean number of molecules in a detection volume is approximately 0.5. A detection volume of the order of 10(-13) L thus gives smaller RSD values for concentrations from 10(-11) to 10(-10) M than does one of the order of 10(-15) L, which is widely used in FCS. This prediction is supported by the molecular simulations, taking into account the finite molecule brightness and background emission. In the second part of the paper, the RSD is evaluated experimentally with an FCS setup with a detection volume of 1.1 x 10(-13) L. The newly developed objective lens, serving as the bottom of the sample cell in this setup, has a large numerical aperture (0.9) without using immersion liquid. When a calibration line was made by 30-s FCS measurements of Cy3-labeled, 112-mer single-stranded DNA solutions, the RSD roughly agreed with the simulation result and was less than 0.1 for DNA concentrations from 2 x 10(-11) to 10(-10) M.

Ultra-slim laminated capillary array for high-speed DNA separation.

We developed a new kind of capillary array for electrophoresis by using the numerical-control (NC) wiring technique conventionally used to produce printed-circuit boards. Laminating two polyimide sheets after laying cylindrical capillaries between them according to designed geometries, we fabricated a 16-lane laminated capillary array (LCA) 9.9 cm long, 7.2 cm wide, and 0.5 mm thick in which the effective length of all capillaries was only 10.9 cm. This compact LCA thus had separation columns as short as those in capillary array electrophoresis chips fabricated by lithography techniques. Like conventional capillary arrays, it also enabled pipetting-less direct injection of analytes from sample preparation plates. Using the LCA with LIF detection and a replaceable fluid sieving matrix, we demonstrated high-speed ssDNA fragment separations. At an electric field strength of 316 V/cm, 15 fragments ranging from 50 to 500 bases were completely separated within 5.8 min in all lanes. The lane-to-lane CV of migration time was only 0.38%, and the fragment size for which the resolution per base was 0.59 was 258 +/- 15 bases (average +/-SD).

High-speed DNA sequencing by tube-based capillary electrophoresis.

We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE.

Integrated on-capillary instrumentation for gene expression measurement directly from cells.

We studied the fundamental instrumental issues relevant to a capillary-based integrated system to measure expression of a specific gene directly from cells. Samples were introduced into a capillary by use of a syringe pump. All reactions were carried out in a microthermocycler, where a part of the capillary having 1 microL inner volume was used as a reaction vessel. First, cells were lysed by heating to release RNA, followed by deoxyribonuclease (DNase) treatment. Then, reverse transcription-polymerase chain reaction (RT-PCR) was performed to obtain amplified products from the targeted mRNA. Finally, the product was verified by capillary electrophoresis (CE) with laser-induced fluorescence detection. The whole protocol was completed in the system in 3 h. PCR product from beta-actin mRNA in 16 human lymphoblast cells was obtained with a signal-to-noise ratio (S/N) of 3400 +/- 730 (n = 3). Therefore, the system is reproducible and sensitive enough to measure gene expression from a single cell. We show that the amplified fragment from breast cancer-specific mRNA was obtained from cells of breast cancer cell line, but was not obtained from cells of hepatoma cell line. These results therefore lay the foundations for future CE or microchip instrumentation for high-throughput automated gene-expression analysis.

Electrophoretic quantitation of nucleic acids without amplification by single-molecule imaging.

We have developed a novel high-performance quantitative assay for unamplified nucleic acids that is based on single-molecule imaging. The apparatus is a simple but highly sensitive single-molecule detection system that uses a normal CCD camera instead of an image-intensified CCD camera. After the DNA molecules in a sample were labeled with YOYO-1, they were induced to migrate electrophoretically in a polymer solution and imaged. No chemical or biochemical amplification was required. Direct quantitation of the sample by counting molecules was possible, because the number counted over the measurement period was directly proportional to the concentration of DNA molecules in the sample. Nonspecifically labeled impurities that would degrade the sensitivity of the assay were successfully reduced and discriminated from the DNA molecules by differences in electrophoretic mobility. By using beta-actin DNA (838 bp) as a model sample, we demonstrate that this protocol was fast (10-min measurement period), highly sensitive (limit of quantitation: approximately 10(3) copies/sample, or 3 x 10(-16) M), quantitative, and covered a wide linear dynamic range (approximately 10(4)). This high-performance assay promises to be a powerful technology for the quantitation of specific varieties of mRNA in the study of gene functions and diseases and in the clinical detection of mutant cells.