Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-19
2003-12-16
Forman, B. J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S174000, C435S283100, C435S091100, C435S091200, C536S024300, C422S068100
Reexamination Certificate
active
06664044
ABSTRACT:
TECHNICAL FIELD
This application is a 371 of PCT/JP98/02389, filed May 29, 1998.
The present invention relates to a method for stably retaining an extremely minute amount of a solution used for a chemical reaction or the like on a surface of a glass plate or the like, a method for effecting a reaction such as PCR using minute amount of reagents, and a reaction vessel suitable for use therein.
BACKGROUND ART
In recent years, reactions such as PCR (polymerase chain reaction) and EIA (enzyme immunoassay) have been utilized in the field of biotechnology. In these reactions, reduction of an amount of a reaction solution to a minute amount is important from the viewpoint of cost reduction or the like. Ink jet technique which deposits ink on paper to visualize letters, characters and images was originally developed for printing. However, ink jet technique is capable of precisely dispencing a minute amount of a liquid, and thus applications thereof have been attempted as a technique for reducing an amount of a reaction solution to a minute amount.
U.S. Pat. No. 4,877,745 and BioTechniques 15, 324 (1993) disclose a method for utilizing ink jet technique as a dispencer is disclosed. U.S. Pat. No. 5,449,754 and U.S. Pat. No. 5,474,796 disclose application of ink jet technique to organic chemical syntheses. Japanese Unexamined Patent Publication No. 262256/1992, Japanese Unexamined Patent Publication No. 289457/1992 and Analytical Chemistry 67, 3051 disclose application of ink jet technique to immunological reactions. For example, Japanese Unexamined Patent Publication No. 262256/1992 discloses a method which comprises preliminary printing a necessary reagent on a film by ink-jetting, adding a sample solution thereto in an amount on the order of 50 &mgr;l (microliter) at the time of use to permit the reagent to dissolve in the solution and to thereby effect reaction, followed by washing and color development to perform detection.
As described above, it has been attempted to utilize ink-jetting as a dispenser for a reaction solution in a chemical reaction. In this connection, when a number of reactions are effected in parallel in a clinical test or the like, it is desired to minimize an amount of each individual droplet, for example, to reduce the amount to 100 nl (nanoliter) or less. However, no cases have been reported where a reaction in a clinical test, for example, PCR or an immunological reaction is effected in an extremely minute amount of a solution of the order of 10 nl to 1 pl (picoliter) by using ink-jetting. In the case of the above-mentioned Japanese Unexamined Patent Publication No. 262256/1992, the amount of the solution in the reaction is 50 &mgr;l. This is not an extremely minute amount.
One of the reasons why no cases have been reported where a reaction in an extremely minute amount is effected using ink-jetting is that an extremely minute amount of a solution dispenced by ink-jetting vaporizes in about several seconds the in air. As in U.S. Pat. No. 5,449,754, with respect to an organic chemical reaction which completes in a moment, examples of a reaction in an extremely minute amount have been reported. However, a reaction in a clinical test generally takes a time period of several minutes to several hours. It is difficult to retain a reaction solution during the period in the air without evaporation of the reaction solution.
Further, when it is detected by fluorometry, colorimetry or the like whether a reaction occurred or not, the detection is difficult in an extremely minute amount of a reaction solution because a length of an optical path in the extremely minute amount of the-reaction solution is markedly shorter, as compared with a case of an ordinary amount of a reaction solution. Accordingly, a contrivance to elongate the length of the optical Ad path at the time of measurement is desired, although this is not indispensable for effecting a reaction in an extremely minute amount.
SUMMARY OF THE INVENTION
The present invention has been made in view of the above-described problems in the conventional techniques. It is, therefore, an object of the present invention to provide a method for stably retaining an extremely minute amount of a solution projected by, for example, ink jet method for a chemical reaction or the like for a long period of time without evaporation of the solution.
It is also an object of the present invention to provide a method for elongating a length of an optical path in order to facilitate optical detection with respect to an extremely minute amount of a solution projected by ink jet method or the like.
It is further an object of the present invention to provide a method for effecting a reaction, such as PCR, using a minute amount of a reagent projected by ink jet method, and a reaction vessel suitably used therein.
In the present invention, the above object is attained in such a manner that a layer of a liquid, such as an oil, which is hardly miscible with an extremely minute amount of a solution (minute droplet) intended to be retained is formed on a substrate such as a glass plate to retain the minute droplet in the liquid layer with the minute droplet in contact with a surface of the substrate. When the minute droplet is aqueous, the liquid layer applied onto the surface of the substrate may be oily. The minute droplet may be shot into the liquid layer applied onto the substrate from the surface of the liquid layer by ink jet technique or the like.
By appropriately selecting viscosity and thickness of the liquid layer formed on the substrate, the minute droplet shot from the surface of the liquid layer is permitted to deposit on the surface of the substrate and stably retained under cover of the liquid layer. The minute droplet is surrounded by the liquid which is substantially immiscible therewith, and thus evaporation thereof can greatly be reduced. Further, since the minute droplet is deposited and fixedly retained on the surface of the substrate, a reagent may further be added to the minute droplet as such by ink jet technique.
For example, when the minute droplet is shot into the liquid layer in an amount of 40 pl by ink jet method, optimum viscosity of the liquid layer is 20 to 50 cp and optimum thickness of the liquid layer is 20 to 30 &mgr;m. If the liquid layer applied onto the surface of the substrate has too small a thickness, the minute droplet partially comes out of the liquid layer and evaporates. On the other hand, if the liquid layer has too large a thickness, the minute droplet is suspended in the liquid layer and positionally unsettled. Accordingly, addition of a reagent thereto or the like is difficult.
The surface of the substrate may have water repellency. The water repellency used herein is expressed as an amount of a rising angle (contact angle) of the droplet, which is deposited on the surface of the substrate, relative to the surface of the substrate. The larger the contact angle, the more water repellent. In the present invention, a surface of a substrate which has a larger contact angle of a droplet than that of a droplet deposited on a commonly used glass substrate is represented as having water repellency. The water repellency may be imparted to the substrate by preparing the substrate itself from a material having water repellecy, such as a polypropylene, or by coating a water repellent material over the substrate.
If the surface of the substrate has water repellency, an aqueous minute droplet deposited on the surface of the substrate has a smaller area of contact with the substrate and thus rises in the thickness direction of the liquid layer. Accordingly, when a chemical reaction which occurs in the minute droplet is detected by an optical method such as fluorometry, colorimetry or the like in the direction perpendicular to the substrate, the detection is facilitated because of the elongation of the length of the optical path. In this case, the substrate having water repellency or water repellent coating required not to affect the optical measurement of the minute droplet.
If the minute droplet is shot into
Forman B. J.
Kenyon & Kenyon
Toyota Jidosha & Kabushiki Kaisha
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