Integrated microvolume device

Abstract

A fully integrated monolithic small volume PCR-CE device in glass, or the like materials, is fabricated using thin film metal heaters and thermocouples to thermally cycle sub-microliter PCR volumes. Successful amplification of a PCR fragment is demonstrated on a PCR-CE chip. The process utilizes a linear polyacrylamide surface coating coupled with addition of BSA to the amplification buffer was necessary to obtain amplification efficiencies comparable to a positive control. The micro-reactor reduced significantly the time required for amplification and the reaction volume was in the sub-microliter regime. Likewise addressed are the known problems connected with reliable microfabrication of metal coatings and the insulating layers required to shield these layers from the PCR reaction mix, and the longstanding unresolved issue of exposed metal regions in the PCR-CE chip resulting in electrolysis of water and bubble formation whenever a voltage is applied. The instant teachings employ external heaters and thermocouples and, as such, have alleviated many of these problems. Heaters and thermocouples may still be thin film deposited after chip bonding allowing for easy scale-up to multichannel devices. In addition, direct deposition of these chip components insures good thermal contact with the PCR reactor.

We claim:  
     
         1 . In a fully integrated glassine monolithic microvolume PCR-CE device, 
 the improvement which comprises: 
 utitilizing thin film metal heating means to maintain a predetermined range of temperature for desired denaturations; and  
 employing thermocoupling means for assuring requisite cycling of sub-microliter PCR volumes.  
   
     
     
         2 . Device as defined in  claim 1 , further comprising a linear polyacrylamide surface coating; and a means for passivating the surface of an involved PCR mix.  
     
     
         3 . Device as defined in  claim 2 , wherein said means for passivating is BSA added to the amplification buffer.  
     
     
         4 . Method of microfabricating the device of  claim 3 , comprising the steps of: 
 providing a means for delivering fluid samples a series of PCR chambers as well as a means for removing the samples from the chambers;    disposing said thin film metal heating means on by thin film deposition; and,    harmonizing thermal cycling and resistance temperature sensing and concomitant adjustments by means of utilizing cycling control software further comprising heating and cooling times as part of a user-set hold time such that the hold time at each temperature does not begin until the actual temperature is within a user-defined percentage of a predetermined set point.    
     
     
         5 . An integrated monolithic PCR reactor coupled to a microcapillary electrophoresis system including an electrophoresis column, the apparatus comprising in combination: 
 a reaction cavity microfabricated within two-bonded substrates defining a space therebetween;    a plurality of apertures functioning as access means for introducing fluids into said reaction cavity;    exit channel means for connecting the reactor to the electrophoresis system and for injecting material onto the electrophoresis column incorporated therein;    a heating means for regulating temperature disposed on the outside of one of said bonded substrates; and,    a thermocouple disposed in a least one position selected from the group consisting of within the space defined by said reaction cavity and between said heater and said bonded substrate.    
     
     
         6 . Apparatus as defined in  claim 5 , said heating means for regulating temperature further comprising a thin film resistive heater.  
     
     
         7 . Apparatus as defined in  claim 5 , said heating means for regulating temperature further comprising a Minko-type contact resistance heater.  
     
     
         8 . Apparatus as defined in  claim 5 , wherein said bonded substrate upon which said means for regulating temperature is disposed is about 1 mm thick or less and said other substrate is thicker.  
     
     
         9 . Apparatus as defined in  claim 5 , heating means for regulating temperature further comprising a heater disposed within said reaction cavity and covered with an insulating layer.  
     
     
         10 . Apparatus as defined in  claim 5 , said thermocouple is Au:Cr type junction.  
     
     
         11 . Apparatus as defined in  claim 5 , wherein said thermocouple is disposed between said heater and said bonded substrate.  
     
     
         12 . Apparatus as defined in  claim 6 , wherein said thermocouple is covered with a dielectric layer.  
     
     
         13 . Apparatus as defined in  claim 6 , wherein said thermocouple is within the space defined by said reaction cavity.  
     
     
         14 . Apparatus as defined in  claim 1 , each said substrate being at least one material selected from the group consisting of soda lime glass, borofloat glass, plastic, PMMA, and the like glassine substances.  
     
     
         15 . Device as defined in  claim 1 , further comprising an oligonucleotide array disposed therein, said oligonucleotide array including a substrate having a plurality of positionally distinct oligonucleotide probes coupled to a surface of said substrate.  
     
     
         16 . Device as defined in  claim 1 , wherein said fully integrated glassine monolithic microvolume PCR-CE device further comprises at least first and second planar members, said first planar member having a first surface and wells disposed in said first surface, said second planar member having a second surface, said second surface being mated to said first surface whereby said wells are contiguous with a respective plurality of cavities having curved insides spaces defining said surface and well-mated arrangement.  
     
     
         17 . Device as defined in  claim 16 , wherein a temperature sensor is deposited on said second surface wherein when said second surface is mated with said first surface, said temperature sensor on said second surface is positioned within said cavity whereby a temperature at said temperature sensor is substantially the same as a temperature within said plurality of cavities.  
     
     
         18 . Device as defined in  claim 5 , wherein said supplemental means for resistance temperature sensing further comprises a thermocouple having a sensing junction positioned adjacent said cavity, and a reference junction positioned outside of said cavity, said thermocouple being electrically connected to a means for measuring a voltage across said thermocouple.  
     
     
         19 . Microfabricated reaction chamber system as defined in  claim 5 , said thermocouple further comprising at least a first gold film adjoined to a chromium film as said sensing junction and said chromium film adjoined to a second gold film as said reference junction.  
     
     
         20 . Device as defined in  claim 3 , further comprising an effective amount of four deoxynucleotide triphosphates, a nucleic acid polymerase and amplification primer sequences disposed within said fully integrated glassine monolithic microvolume PCR-CE device.