Patent Application
20120074108
The present invention relates to a method for making a micro-hole structure, and in particular to a method for making an hourglass shape micro-hole structure with a reflow section and a structure thereof.
Heretofore, the fabrication of a micro-hole that traps and positions a cell is generally carried out with micro-machining, such as micro-electro-mechanical system (MEMS), which is a photolithographic technique employing micro-manufacturing processes of exposure, development, and etching for a micro-opening of the size of a cell. Such a process is disadvantageous of using expensive facility and being complicated and consuming labor and time.
An example is U.S. Pat. No. 6,699,896, which discloses a fabrication method for making a planar microelectrode for measuring ion channel of a cell membrane, featuring adoption of photolithography in combination with molding of polymer PDMS (poly-dimethylsiloxane) to realize efficient formation of tiny holes for trapping cell and forming high resistance to measure cell-membrane surface ion-channel signals. Another example is U.S. Pat. No. 6,776,896, which discloses a method for positioning cells for the measurement of electrophysiological signal of the cells, featuring formation of a flow field around an aperture with a structural design assisted by a perfusion system to facilitate movement of a cell toward the aperture thereby realizing precise positioning of the cell on the aperture.
Laser drilling is commonly employed in the industry, which uses high energy carried by excitation of laser to spontaneously melt a material for forming a hole. This is an effective and efficient manner for fabrication of micro holes.
T. Sordel et al. discloses in a literature of “Hourglass SiO2 coating increases the performance of planar patch-clamp,” published in J Biotechnology 125 (1), 2006, that the hourglass SiO2 coating enhances the performance of planar patch clamp chip. The straight hole is essentially fabricated by standard photolithography on silicon substrate. SiO2 is coated on the pore to form an hourglass structure by plasma-enhanced chemical vapor deposition (PECVD). The reference proves that the hourglass feature performs better seal quality.
K. G Klemic et al. discloses in Pflugers Arch Eur J Physiol 449 (6), 564-572 an air-molding technique to create a micro-hole on polydimethylsiloxane (PDMS) membrane. A metal plate with a 2 μm through hole is firstly defined. PDMS is then coated on the metal plate which the air forces on the hole to penetrate the PDMS membrane and is cured rapidly during the air blowing. The size of the aperture is defined by the metal through hole. This is an economic fabrication method, but it needs sophisticated operation. The aperture presents a flat surface that contact with cell during patch-clamp experiment.
Also, T. Lehnert et al. shows how hollow SiO2 micronozzles for electrical measurements on living cells are realized in Applied Physics Letters 81 (26), P. 5063-5065. It demonstrates a fabrication method to generate a hollow SiO2 micronozzle to mimic the traditional micropipette pressing against the cell membrane. The fabrication comprises a series of dry etching and deposition procedures. The result shows that the chip is unable to obtain gigaseal.
A literature published in Microfluidics and Nanofluidics 3 (1), P. 109-117 discloses that a phosphosilicate glass (PSG) reflow is used to form a 3-dimensional micro-aperture. A series of wet etching and dry etching is performed to create a funnel-liked aperture. PSG is then deposit on the aperture surface and heated to reflow to obtain very smooth surface. The opening of the aperture is quite similar to our hourglass-shaped aperture. The result also demonstrates the capability of gigaseal formation. However, the fabrication process is complicated and costly.
In the 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences held at Boston, Mass., USA held at Oct. 9-13, 2005, Levent Yobas et al. present a fabrication method of lateral glass capillaries for patch clamping. It utilizes keyhole-void formation and thermal-reflow of phosphosilicate glass (PSG) in silicon trenches to fabricate lateral glass capillaries. The chip allows optical observation during patch-clamp experiment to optimally choose good cells for patch. Hence the gigaseal rate is quite good, up to 60%.
Also, Adrian Y. Lau et al. presents in the 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences about the raised lateral patch clamp array in open-access fluidic system. PDMS is a widely used material for microchannel fabrication. The presentation demonstrates the usage of microchannel opening as cell patch site for ion channel recording. The opening is a 2×2 μm square. This is a cheap and simple fabrication method, but it is very difficult to have gigaseal formation.
Biophysical Journal 82 (6), P. 3056-3062 in 2002 publishes an article of “Whole cell patch clamp recording performed on a planar glass chip” of N. Fertig et al. Quartz is chosen as substrate due to its excellent electrical properties. The micro-aperture is generated by wet etching and ion track etching. The aperture presents a flat surface that contacts with cell during patch-clamp experiment. Gigaseal is proven capable.
R. Pantoja et al. publishes the usage of photo-lithographical process to fabricate micro-apertures on silicon chips in Biosensors and Bioelectronics 20 (3) of 2004, P. 509-517. Double side dry etching is performed to obtain 0.7-2 μm aperture. The aperture also presents a flat surface.
K. Schroeder publishes an article in Journal of Biomolecular Screening 8 (1), P. 50-64 in 2003. The patch clamp chip in this article is fabricated by laser drilling technique on polyimide. The price could be quite cheap, but the chip could not provide gigaseal formation, which limits its application on measuring precise ion channel activities.
Another paper presented in 9th International Conference on Miniaturized Systems for Chemistry And Life Sciences held at Boston, Mass., USA at Oct. 9-13, 2005 by A. Minamino et al is about the fabrication of micropipette chips for simultaneous electrophysiological and optical measurements. Photo-lithographical process is used to fabricate micro-apertures on silicon chips. Double side dry etching is performed to obtain 0.5-2 μm aperture. The aperture also presents a flat surface.
An example is shown in U.S. Pat. No. 4,948,941, which discloses a method of laser drilling that produces a uniform hole in a substrate and features placing a sacrificial member over the substrate, whereby the laser drilling forms a tapering portion of a drilled hole only in the sacrificial member and the portion of the drilled hole formed in the substrate is a micro-hole that maintains substantially uniform. The sacrificial member is then removed and a micro-hole of substantially uniform dimension is thus obtained. U.S. Pat. No. 6,642,477 discloses a method for laser drilling that produces a counter-tapered micro-hole, featuring applying regular laser drilling to form a tapered micro-hole in a substrate and then displacing and rotating the substrate to change the drilling site and laser incidence angle for forming a counter-tapered micro-hole. Another example is U.S. Pat. No. 7,019,257, which discloses a laser drilling method for forming a micro-hole that is comprised of an end of a counter-tapered shape and an end of cylindrical shape, featuring first applying regular laser drilling to form a uniform micro-hole of a suitable size in a substrate and then effecting displacement and rotation of the substrate, with precisely calculated angle and location of laser incidence to thereby form a micro-hole of a counter-tapered shape.
However, those conventional techniques are only useful in forming micro-holes of conic (tapering) or cylindrical shape and it is in general difficult to form a hole of a specially-configured cross section with the conventional techniques.
Therefore, an objective of the present invention is to provide a micro-hole structure and a manufacturing method therefor for making a micro-hole having a unique configuration.
To achieve the above objective, in accordance with the present invention, a substrate having first and second surfaces is provided, and a working energy source carrying a predetermined working energy is projected onto a predetermined drilling site on the first surface of the substrate.
In a predetermined period of time of projection of the working energy source, the working energy source melts a portion of the substrate at the drilling site and defines a first opening in the first surface of the substrate and further penetrates into the substrate to form a working energy entering section in the substrate next to the first surface until the working energy source penetrates through the second surface of the substrate thereby forming a micro-hole that extends through both the first and second surfaces of the substrate.
In the micro-hole, a portion of the substrate next to the second surface that is melted by the working energy source is in a molten condition and reflows in a direction opposite to the projection of the working energy source to thereby form a reflow section in the micro-hole next to the second surface of the substrate, also defining a second opening in the second surface of the substrate, and forming a through hole section between the working energy entering section and the reflow section. The reflow section extends from the second opening to and communicates the through hole section and has a bore diameter converging from the second opening toward the through hole section.
The method of the present invention allows for fabrication of a micro-hole smaller than 10 micrometers and having an unsymmetrical hourglass like cross-section. Such a configuration is well suitable for trapping and positioning a cell for measurement of ion channel of cellular membrane with substantially increased rate of success. The method of the present invention is economic and efficient and remarkably reduces manufacturing costs. In addition, the micro-hole structure in accordance with the present invention can be arranged in an array or can be integrated with a micro-fluidic system to provide an efficient drug screening platform.
Further, the present invention also features the adoption of two or more stages emission of laser pulses as a working energy source for forming a micro-hole in a substrate to be machined whereby precise control of the bore diameter can be realized at the time when a reflow section of the micro-hole is being formed in the substrate next to a second surface of the substrate.
The present invention will be apparent to those skilled in the art by reading the following description of the best mode for carrying out the present invention, with reference to the attached drawings, in which:
With reference to the drawings and in particular to
The optic operation mechanism 1 comprises a first reflector carrier 11, a second reflector carrier 12, a third reflector carrier 13, a fourth reflector carrier 14, a first support member 15, a second support member 16, a third support member 17, and a working stage 18. The first reflector carrier 11 is arranged at a lower end of the first support member 15, while the second reflector carrier 12 is arranged at an upper end of the first support member 15. The third reflector carrier 13 is coupled to a support-member coupling end 171 of the third support member 17 and the fourth reflector carrier 14 is coupled to a carrier coupling edge 172 of the third support member 17. The third support member 17 is coupled to a support-member coupling edge 161 of the second support member 16. The working stage 18 is coupled to a stage coupling edge 151 of the first support member 15.
The laser beam modulation lens set 2 comprises a first reflector 21, a second reflector 22, a third reflector 23, a fourth reflector 24, and a focus lens 25. The first reflector 21 is arranged in the first reflector carrier 11. The second reflector 22 is arranged in the second reflector carrier 12. The third reflector 23 is arranged in the third reflector carrier 13. The fourth reflector 24 is arranged on a reflection end 141 of the fourth reflector carrier 14, while the focus lens 25 is arranged on a focus end 142 of the fourth reflector carrier 14.
Referring to
Referring to
Thereafter, the working energy source generator 4 is properly set to face the first reflector 21. In the instant embodiment, a 25 W CO, laser device is employed to serve as the working energy source generator 4 and the operation of the laser device requires two different inputs, one being continual small pulse signals, which function to pre-ionize the carbon dioxide gas to facilitate the excitation of laser beam and the other being a pulse-width modulated (PWM) command, which functions to excite the laser beam for effecting hole drilling on the substrate 3. In the instant embodiment, the continual small pulse signals include signals of 5 KHz, 5 Vpp, and 0.5% duty cycle. The PWM command includes signals of 400 Hz, 5 Vpp, and 3.6% duty cycle. The drilling operation is carried out with signals of five durations.
The positions of the laser beam modulation lens set 2 and the working stage 18 are then adjusted. Since the fourth reflector carrier 14, the third support member 17, and the working stage 18 are interfaced by a stepping motor to a computer device, an operator may operate the computer device to control the stepping motor for moving the working stage 18 up and down along the stage coupling edge 151 of the first support member 15 in a direction of Z-axis to effect adjustment of the distance between the focus lens 25 and the working stage 18 and thus set a proper focusing distance for the laser beam. Thus, the laser beam can be made focused on a focusing spot of around 1-500 μm on the first surface 31 of the substrate 3.
Further, the operator may use the computer device to control the stepping motor so that the fourth reflector carrier 14 is driven to move back and forth along the carrier coupling edge 172 of the third support member 17 in the direction of X-axis and also the third support member 17 is driven to move back and forth along the support-member coupling edge 161 of the second support member 16 in the direction of Y-axis to thereby define various drilling sites on the first surface 31 of the substrate 3, where hole drilling is to be effected, thereby allowing formation of a micro-hole array. This can be integrated with micro-fluidic systems for applications in high throughput screening, and is also applicable to distribution of fluid, such as forming a micro-nozzle with the unique configuration of the micro-hole in accordance with the present invention.
Once the positions of the laser beam modulation lens set 2 and the working stage 18 are properly set, hole drilling is carried out. Since the instant embodiment uses a laser generator to serve as the working energy source generator 4, manipulation of laser excitation signal allows excitation of the working energy source generator 4 to generate a working energy source 41 for a predetermined period of time. The working energy source 41 includes a laser beam of having predetermined number, duration, and frequency of laser pulse. The working energy source 41 is reflected to the second reflector 22 by the first reflector 21 and further reflected by the second reflector 22 toward the third reflector 23, followed by reflection by the third reflector 23 toward the fourth reflector 24 and further reflection by the fourth reflector 24 toward the focus lens 25, which then focuses the working energy source 41 to form a focused laser beam 42 projecting to a selected drilling site on the first surface 31 of the substrate 3.
The focused laser beam 42 then causes melting of the material of the substrate 3 at the selected drilling site on the first surface 31 of the substrate 3, thereby forming a first opening 51 in the first surface 31 of the substrate 3. Proceeding application of the focused laser beam 42 to the substrate 3 forms, in the substrate 3, a working energy entering section 54 that commences under the first surface 31 of the substrate 3, until the focused laser beam 42 just penetrate through the second surface 32 of the substrate 3. As such, the micro-hole structure 5 that extends through both the first surface 31 and the second surface 32 of the substrate 3 is made.
Thereafter, as shown in
The working energy entering section 54 extends from the first opening 51 to the internal through hole section 53 and the bore diameter of the working energy entering section 54 is convergent from the first opening 51 toward the through hole section 53. The second opening 52 is substantially aligned with and in communication with the first opening 51. The internal through hole section 53 is close to the second opening 52. With the first opening 51, the second opening 52, and the internal through hole section 53 respectively having a first opening diameter d1, a second opening diameter d2, and a through hole section diameter d3, the through hole section diameter d3 is less than the first opening diameter d1 and is also less than the second opening diameter d2. The through hole section 53 is located closer to the second opening 52 than the first opening 51. Thus, the micro-hole structure 5 formed with the above described reflowing process is of an unsymmetrical hourglass shape.
In the instant embodiment, the second opening diameter d2 so formed is around the size of a cell, about 20-30 μm and is thus well applicable to trapping and positioning of a cell. The size of the through hole section diameter d3 is substantially identical to that of a micropipette used in known patch-clamp techniques, about 2-10 μm, and is thus well suitable for study of electrophysiological response of ion channel of the cellular membrane. The unique hourglass configuration of the present invention facilitates smooth introduction of a cell into the first opening 51 of the micro-hole 5 and also enhances seal resistance between the cell and the glass material of the substrate up to even 109 ohms. This allows the measurement of the tiny current induced by movement of ions through the cell surface.
The through hole section diameter d3 formed in accordance with the present invention is of a size about 500 nanometers to 200 micrometers. It is noted that to fabricate a through hole section having a diameter between 500 nanometers to 200 micrometers in a substrate 3 of a thickness of 0.01-10 millimeters, the above-discussed working energy source 41 (which in the instant embodiment includes a laser beam having a predetermined power) carrying a predetermined power is used for a predetermined period of time of emission. Parameters that affect the emission of the laser energy include number, duration, and frequency of laser pulses. For example, the number of laser pulses can be set in a range of 1-1010, and the pulse duration and pulse frequency are adjusted accordingly. The period of time of radiation is set in accordance with the thickness and material of the substrate 3 and the size of the micro-hole to be fabricated. Besides the gas laser beam generator mentioned above, the working energy source generator 4 can also be a solid laser beam generator or a semiconductor laser beam generator; the working energy source generator 4 can be a pulse laser beam generator or a continuous laser beam generator.
Referring to
Firstly, a substrate of a predetermined thickness is provided (step 101). The substrate has a first surface and a second surface. The substrate is then positioned on a working stage (step 102).
A working energy source generator is then used to irradiate a working energy source of a predetermined working energy for a predetermined period of time (step 103). The working energy source is projected through a laser beam modulation lens set onto a predetermined drilling site on the first surface of the substrate (step 104). The working energy source projected onto the first surface of the substrate may either carry a fixed working energy or a gradually decreased working energy.
The working energy source melts the material of the substrate at the drilling site on the first surface to form a working energy entering section in the substrate next to the first surface of the substrate, until the working energy source penetrates through a second surface of the substrate (step 105). As a consequence, a micro-hole is formed through both the first surface and the second surface of the substrate at the predetermined drilling site on the first surface of the substrate (step 106).
The portion of the material of the substrate at the micro-hole adjacent to the second surface of the substrate, which is in a molten condition due to be melted by the working energy source reflows in a direction opposite to the incidence of the working energy source (step 107), thereby forming a reflow section of the micro-hole in the substrate next to the second surface and defining a second opening in the second surface of the substrate, and thus forming a through hole section in the junction between the working energy entering section and the reflow section (step 108), with the reflow section extending from the second opening to and in communication with the through hole section and having a bore diameter converging from the second opening to the through hole section.
In the instant embodiment of
In
A third example is shown in
In the above three examples, all the pulses in the first and second stage laser pulse sequences have identical power. In the fourth example shown in
Although two-stage emission of laser pulses is used in the present invention, it is apparent that the number of stages of emission of laser pulses can be varied in accordance with practical applications. In other words, two or more stages emission of laser pulses can be used.
Referring simultaneously to
A working energy source generator is then used to emit a stage laser pulse sequence S1 of first stage laser pulses serving as a first stage working energy source for a predetermined period of time (step 103a). The working energy source is projected through a laser beam modulation lens set onto a predetermined drilling site on the first surface of the substrate (step 104).
The working energy source melts the material of the substrate at the drilling site on the first surface to form a working energy entering section in the substrate next to the first surface of the substrate, until the working energy source penetrates through a second surface of the substrate (step 105). As a consequence, a micro-hole is formed through both the first surface and the second surface of the substrate at the predetermined drilling site on the first surface of the substrate (step 106).
Then, a second stage laser pulse sequence S2 of second stage laser pulses, serving as a second stage working energy source, is projected to the micro-hole. At this time, the portion of the material of the substrate at the micro-hole adjacent to the second surface of the substrate, which is in a molten condition due to be melted by the second stage working energy source, reflows in a direction opposite to the incidence of the second stage working energy source (step 107a), thereby forming a reflow section of the micro-hole in the substrate next to the second surface and defining a second opening in the second surface of the substrate, and thus forming a through hole section in the junction between the working energy entering section and the reflow section (step 108), with the reflow section extending from the second opening to and in communication with the through hole section and having a bore diameter converging from the second opening to the through hole section
The working energy contained in the second stage laser pulse sequence S2 is different from that of the first stage laser pulse sequence S1, whereby precise control of the bore diameter can be realized at the time when the reflow section of the micro-hole is being formed in the substrate next to the second surface. In the above embodiments, the working energy contained in the second stage laser pulse sequence S2 is less than that of the first stage laser pulse sequence S1.
Although the present invention has been described with reference to the preferred embodiments thereof, as well as the best mode for carrying out the present invention, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.
This application is a Divisional patent application of co-pending application Ser. No. 12/230,819, filed on 5 Sep. 2008, now pending. The entire disclosure of the prior application Ser. No. 12/230,819, from which an oath or declaration is supplied, is considered a part of the disclosure of the accompanying Divisional application and is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12230819 | Sep 2008 | US |
| Child | 13311620 | US |
