A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability

Abstract

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-inanswer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 l of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.full integration ͉ micro total analysis system ͉ microdevice ͉ pumping ͉ valving T he next revolution in personalized medicine, forensic science, and biowarfare defense will be impelled by analysis systems that provide a quantum leap in terms of functionality, time to result, and cost effectiveness. These systems need to meet several requirements, including a design conducive with low-cost manufacturing, turn-key operation with fast analysis times, and the ability to manipulate small volumes from crude samples. One example is the micrototal analysis system (-TAS) described conceptually more than a decade ago by Manz et al. (1). Prophetically, they stated that, ''. . . the detector or sensor in a TAS does not need high selectivity, because the sample pretreatment serves to eliminate most of the interfering chemical compounds.'' There are multiple examples in the literature of steps taken toward the advancement of integrated microfluidic genetic analysis (MGA) systems (refs. 2-4; also see ref. 5 for a comprehensive review); however, after a decade and a half, no bona fide microfluidic device has been presented that is capable of nanoliter flow control and integration of an electrophoretic separation with comprehensive sample pretreatment (DNA purification and PCR amplification).The MGA system described in this report brings together many advances in microfluidics over the last decade, exploiting differential channel flow resistances (6), elastomeric valves (7, 8), laminar flow (9), and electrophoretic mobility within the device, in concert with external fluid flow control from a syringe pump for sample and reagent delivery. Nucleic acid purification through solid-phase e...

Theory, fabrication and applications of microfluidic and nanofluidic biosensors

“…Patterns defined in lithography are physically transferred or transcribed into the substrate using wet or dry etching. Wet etching can be performed using hydrofluoric acid (HF) [31,60] or buffered oxide etch [6] for glass and KOH for silicon, respectively. HF gives isotropic wet etching thus undercutting the masking layer and resulting in semicircular sidewalls or sidewalls that taper downward.…”

“…For example, a polymer-based disposable device capable of detecting 0.25-10 ppm of anaesthetic propofol within 60 s has been already demonstrated (figure 5) [114]. Poly(dimethyl siloxane) (PDMS) is the most commonly used polymer for fabrication of fluidic channels [3,4,11,60] towards biosensor applications though poly(methyl methacrylate) (PMMA) is also used [115]. Conducting polymers such as poly(phenylene vinylene) [23], and its soluble derivatives along with polyaniline have been used for electronic components [28].…”

“…Specifically, the review will discuss the underlying physical and operating principles, as well as the fabrication of biosensor devices that rely on microfluidics and nanofluidics. There has been a growing desire to create portable detection devices that can perform measurements in a matter of minutes using increasingly smaller sample volumes [10,24,25,[60][61][62] approaching the picolitre range. Several commercial examples for biosensing platforms exist including point-of-use type kits such as home pregnancy tests for detection of hormones such as human chorionic gonadotropin (hCG) for early indication of pregnancy.…”

“…Typically PDMS devices are bonded to glass covers by placing the glass cover in contact with the PDMS and curing the PDMS while applying pressure [60,131] or by simply applying pressure [122]. PDMS can also be used as an intermediate layer for glass-to-glass bonding.…”

High‐resolution electrophoretic separation and integrated‐waveguide excitation of fluorescent DNA molecules in a lab on a chip

“…Microfluidic (MF) lab-on-a-chip (LOC) devices [1,2] have enabled integrated DNA sequencing [3] and genetic diagnostics [4,5]. Capillary electrophoresis (CE) separation is a powerful method over a broad range of molecular sizes, thanks to flexible MF protocols, exploiting a variety of sieving gel matrices.…”