Unitary Biochip Providing Sample-in to Results-out Processing and Methods of Manufacture

Abstract

A biochip for the integration of all steps in a complex process from the insertion of a sample to the generation of a result, performed without operator intervention includes microfluidic and macrofluidic features that are acted on by instrument subsystems in a series of scripted processing steps. Methods for fabricating these complex biochips of high feature density by injection molding are also provided.

What is claimed is: 
     
         1 . A stationary biochip for conducting a multiplexed PCR analysis of a nucleic acid solution, and at least 25 total processing steps, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 2 primer pairs;   at least one fluidic transport channel comprising an inlet for receiving said nucleic acid solution, and a primary flow path, said path in fluidic communication with said at least one reconstitution chamber,   such that when the nucleic acid solution is introduced into said inlet, said solution travels along said flow path to the at least one of the reconstitution chambers thereby reconstituting said lyophilized multiplexed PCR reagent; and   wherein said biochip performs at least 25 total processing steps.   
     
     
         2 . A stationary biochip for conducting a multiplexed PCR analysis of a nucleic acid solution, and at least 25 total processing steps, said biochip comprising a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 7 primer pairs;
 at least one fluidic transport channel comprising an inlet for receiving said nucleic acid solution, and a primary flow path, said path in fluidic communication with said at least one reconstitution chamber,   such that when the nucleic acid solution is introduced into said inlet, said solution travels along said flow path to the at least one of the reconstitution chambers thereby reconstituting said lyophilized multiplexed PCR reagent. and   wherein said biochip performs at least 25 total processing steps.   
     
     
         3 . A stationary biochip for conducting a multiplexed PCR analysis of a nucleic acid solution, and at least 25 total processing steps, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 16 primer pairs;   at least one fluidic transport channel comprising an inlet for receiving said nucleic acid solution, and a primary flow path, said path in fluidic communication with said at least one reconstitution chamber,   such that when the nucleic acid solution is introduced into said inlet, said solution travels along said flow path to the at least one of the reconstitution chambers thereby reconstituting said lyophilized multiplexed PCR reagent; and   wherein said biochip performs at least 25 total processing steps.   
     
     
         4 . A stationary biochip for conducting a multiplexed PCR analysis of a nucleic acid solution, and at least 25 total processing steps, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 80 primer pairs;   at least one fluidic transport channel comprising an inlet for receiving said nucleic acid solution, and a primary flow path, said path in fluidic communication with said at least one reconstitution chamber,   such that when the nucleic acid solution is introduced into said inlet, said solution travels along said flow path to the at least one of the reconstitution chambers thereby reconstituting said lyophilized multiplexed PCR reagent; and   wherein said biochip performs at least 25 total processing steps.   
     
     
         5 . The biochip of  claim 1  or  2  or  3  or  4 , comprising a second reconstitution chamber, said second reconstitution chamber further comprising lyophilized sequencing reagent, and a second inlet on said fluidic transport channel downstream from said first inlet, such that when the reconstituted PCR reagent travels along said flow path to said second inlet thereby reconstituting sequencing reagent. 
     
     
         6 . The biochip of  claim 1  further comprising at least one fluid transport channel and a chamber adapted for at least one of the group consisting of nucleic acid extraction; cell lysis; cell separation; differential cell lysis; differential filtration; total nucleic acid purification; DNA purification; RNA purification; mRNA purification; protein purification; pre-nucleic acid amplification cleanup; single nucleotide polymorphism analysis; VNTR analysis; RFLP analysis; post-nucleic acid amplification cleanup; pre-nucleic acid sequencing cleanup; Sanger sequencing; pyrosequencing and single molecule sequencing; post-nucleic acid sequencing cleanup; reverse transcription; pre-reverse transcription cleanup; post-reverse transcription cleanup; nucleic acid ligation; nucleic acid hybridization; electrophoretic separation and detection; immunoassay; binding assay; protein assay; enzymatic assay; mass spectroscopy; nucleic acid quantification and protein quantification. 
     
     
         7 . A stationary biochip for conducting a multiplexed PCR analysis of a DNA solution, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 7 primer pairs;   at least one fluidic transport channel comprising an inlet for receiving said nucleic acid solution, and a primary flow path, said path in fluidic communication with said least one reconstitution chambers,   such that when the DNA solution is introduced into said inlet, and travels along said flow path to the at least one reconstitution chambers, thereby reconstituting said lyophilized multiplexed PCR reagent.   
     
     
         8 . The biochip of  claim 7  wherein the lyophilized reagent containing at least 7 primer pairs performs Real-time PCR; reverse transcription PCR; asymmetric PCR; nested PCR; LATE PCR; touchdown PCR; digital PCR; rolling circle amplification; strand displacement amplification; multiple displacement amplification; STR amplification; Y-STR amplification; and mini-STR amplification; 
     
     
         9 . A stationary biochip for conducting a multiplexed PCR analysis of a DNA solution, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 16 primer pairs;   at least one fluidic transport channel comprising an inlet for receiving said nucleic acid solution, and a primary flow path, said path in fluidic communication with said least one reconstitution chambers,   such that when the DNA solution is introduced into said inlet, and travels along said flow path to the at least one reconstitution chambers, thereby reconstituting said lyophilized multiplexed PCR reagent.   
     
     
         10 . The stationary biochip of  claim 9  in which the 16 primer pairs are primer pairs for human STR loci. 
     
     
         11 . A stationary biochip for generating a sizing profile from a multiplexed PCR analysis of a nucleic acid solution, said biochip comprising
 a plurality of chambers on a fluidic transport channel, comprising   at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 2 primer pairs, and at least one PCR chamber; and   said fluidic transport channel further comprising a first inlet for receiving said nucleic acid solution into said reconstitution chamber where it mixes with lyophilized reagent to become reconstituted multiplex PCR product, which is delivered via a first outlet to a second inlet into said PCR chamber whereupon amplified multiplexed PCR product is generated and delivered to a second outlet on said fluidic transport channel, whereupon   the amplified multiplexed PCR product is driven to the fluidic transport channel to be subjected to at least one scripted processing step prior to electrophoretic separation such that the products of the at least two primer pairs are fractionated by size.   
     
     
         12 . The biochip of  claim 11  which additionally comprises a separation and detection subassembly. 
     
     
         13 . The biochip of  claim 11  or  12  in which said reconstitution chamber is also the PCR chamber. 
     
     
         14 . The biochip of  claim 11  or  12  or  13  comprising a second reconstitution chamber, said second reconstitution chamber further comprising lyophilized sequencing reagent, and a second inlet on said fluidic transport channel downstream from said first inlet, such that the reconstituted PCR reagent travels along said flow path to said second inlet thereby reconstituting sequencing reagent prior to electrophoretic separation. 
     
     
         15 . A stationary biochip for conducting fragment sizing analysis of an amplified multiplexed PCR solution, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized Internal Lane Sizing Standard reagent, at least one fluidic transport channel comprising an inlet for receiving said multiplexed PCR solution, and a primary flow path, said path in fluidic communication with the at least one of the plurality of chambers containing the lyophilized Internal Lane Sizing Standard reagent   wherein said biochip performs at least 25 total processing steps, and in use, the multiplexed PCR solution is introduced into said inlet, and travels along said flow path to the at least one of the chambers in said plurality of chambers containing the lyophilized Internal Lane Sizing Standard reagent, thereby reconstituting said Internal Lane Sizing Standard.   
     
     
         16 . A stationary biochip for conducting fragment sizing analysis of an amplified multiplexed PCR solution, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized Internal Lane Sizing Standard reagent, at least one fluidic transport channel comprising an inlet for receiving said multiplexed PCR solution, and a primary flow path, said path in fluidic communication with the at least one of the plurality of chambers containing the lyophilized Internal Lane Sizing Standard reagent   wherein the amplified multiplexed PCR solution is introduced into said inlet, and travels along said flow path to the at least one of the chambers in said plurality of chambers containing the lyophilized Internal Lane Sizing Standard reagent, thereby reconstituting said lyophilized the lyophilized Internal Lane Sizing Standard reagent.   
     
     
         17 . A stationary biochip for generating a sizing profile from a multiplexed PCR analysis of a nucleic acid solution, said biochip comprising
 a plurality of chambers on a fluidic transport channel, comprising   at least one first reconstitution chamber, said first reconstitution chamber further comprising at least one lyophilized multiplexed PCR reagent containing at least 2 primer pairs, and at least one PCR chamber; and   at least one PCR chamber; and   at least one second reconstitution chamber, said second reconstitution chamber further comprising at least one lyophilized Internal Lane Sizing Standard reagent;   said fluidic transport channel further comprising a first inlet for receiving said nucleic acid solution into said first reconstitution chamber where it reconstitutes lyophilized PCR reagent cake to become reconstituted multiplex PCR product, and a second inlet for receiving said reconstituted multiplex PCR product into said PCR chamber whereupon amplified multiplexed PCR product is generated, and a third inlet for receiving said amplified multiplexed PCR solution into said second reconstitution chamber where it reconstitutes lyophilized Internal Lane Sizing Standard cake to become reconstituted Internal Lane Sizing Standard reagent/amplified multiplexed PCR solution.   
     
     
         18 . The biochip of  claim 17  whereupon the reconstituted Internal Lane Standard/amplified multiplexed PCR solution is driven downstream in said fluidic transport channel to be subjected to at least one scripted processing step prior to electrophoretic separation such that the products of the at least two primer pairs are fractionated by size. 
     
     
         19 . A stationary biochip for conducting fragment sizing analysis of an allelic ladder, and at least 25 total processing steps, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized Allelic Ladder reagent;   at least one fluidic transport channel comprising an inlet for receiving a liquid, and a primary flow path, said path in fluidic communication with said at least one reconstitution chamber, such that when the liquid is introduced into said inlet, and travels along said flow path to the at least one of the chambers in said plurality of chambers containing the lyophilized Allelic Ladder reagent, thereby reconstituting said Allelic Ladder; and   wherein said biochip performs at least 25 total processing steps,   
     
     
         20 . A stationary biochip for conducting fragment sizing analysis of an allelic ladder, said biochip comprising
 a plurality of chambers, comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized allelic ladder reagent, at least one fluidic transport channel comprising an inlet for receiving a liquid, and a primary flow path, said path in fluidic communication with the at least one of the plurality of chambers containing the lyophilized allelic ladder reagent   wherein the liquid is introduced into said inlet, and travels along said flow path to the at least one of the chambers in said plurality of chambers containing the lyophilized allelic ladder reagent, thereby reconstituting said allelic ladder.   
     
     
         21 . The biochip of  claim 20  whereby said liquid is a solution comprising an Internal Lane Standard. 
     
     
         22 . The biochip of  claim 20  in which the reconstituted ILS is derived from a lyophilized ILS reagent present in a chamber on said biochip. 
     
     
         23 . A stationary biochip for purifying nucleic acids from a cellular lysate, and for performing at least 25 total processing steps, said biochip comprising
 a plurality of chambers comprising at least one reconstitution chamber, said reconstitution chamber further comprising at least one lyophilized Proteinase reagent,   at least one fluidic transport channel comprising an inlet for receiving cellular lysate, and a primary flow path, said path in fluidic communication with said at least one reconstitution chamber, such that when the cellular lysate is introduced into said inlet and travels along said flow path to the at least one of the chambers in said plurality of chambers containing the lyophilized proteinase reagent, thereby reconstituting said lyophilized proteinase reagent, and wherein said biochip performs at least 25 total processing steps,   
     
     
         24 . A biochip for conducting a multiplexed PCR analysis of a sample, said biochip comprising a plurality of chambers on a fluidic transport channel, comprising
 a first chamber comprising a lyophilized proteinase reagent   a second chamber comprising a lyophilized multiplexed PCR reagent containing at least 2 primer pairs;   a third chamber comprising a lyophilized ILS reagent; and   a fourth chamber comprising a lyophilized Allelic ladder reagent;   said fluidic transport channel having a primary flow path and a secondary flow path, said secondary path having an inlet into said fourth chamber; for receiving a second solution into said fourth chamber where lyophilized allelic ladder reagent is reconstituted;   said primary flow path further comprising a first inlet for receiving a nucleic acid solution into said first chamber where proteinase reagent is resuspended and used to lyse cells within said sample to generate a solution containing nucleic acids; and a second inlet for receiving said nucleic acid solution into said second chamber whereupon amplified multiplexed PCR solution is generated, and a third inlet for receiving said amplified multiplexed PCR solution into said third chamber where it mixes with said lyophilized Internal Lane Sizing Standard reagent, thereby reconstituting the lyophilized Internal Lane Sizing Standard reagent; and in parallel, said second solution reconstituting the Allelic ladder reagent, whereupon said resuspended Allelic ladder reagent solution and said resuspended lyophilized Internal Lane Sizing Standard reagent are subjected to electrophoretic separation such that the products of the multiplexed PCR/ILS solution and the Allelic Ladder solution are fractionated by size.   
     
     
         25 . The biochip of  claim 24  further comprising a separation and detection subassembly.