Central to research into cell survival, growth and differentiation in normal and diseased states is the ability to quantify altered patterns of gene expression. Oligonucleotide (1
) and cDNA (3
) hybridization microarrays have emerged as the leading quantitative tool for analyzing transcription of many thousands of genes in a sample simultaneously (4
) yet have known limitations in analytical performance and sample throughput (5
). Real time or quantitative polymerase chain reaction (qPCR) (8
) is the superior alternative because of its high accuracy, precision and dynamic range and, as a consequence, is the reference assay for calibration and validation of microarray data (9
). However, scaling qPCR to analyze larger numbers of genes and samples simultaneously is intrinsically prohibited by the logistics and cost of the assay in its current microliter format in 96- or 384-well microplates.
High throughput PCR strategies have focused on smaller reaction volumes and follow one of two fluidics methods. Fast sequential analysis is exemplified by monolithic, functionally integrated lab-on-a-chip devices that flow a sample bolus through fixed temperature zones of a micromachined channel for target sequence PCR amplification, followed by sequence specific capture by hybridization and electrochemical detection (10
), fluorescence detection (11
) or electrophoretic separation with fluorescent detection (12
). With quantitative performance similar to a microarray, detection sensitivity is further constrained by sample throughput and the increased potential for cross-contamination from processing samples in a common microchannel.
Many of these problems are mitigated in a parallel fluidics approach. Miniaturized versions of microplates based on high-density arrays of wells etched in a planar substrate is the basis for nanoliter- (13
) or picoliter-scale PCR (17
) in an array format. Other embodiments include PCR in microdroplets on a patterned hydrophobic–hydrophilic surface (18
) or in a 2D array of communicating microchannels (19
). Reports of quantitative nucleic acid measurement in these devices have focused on limiting dilution schemes (22
), which is clearly not high-throughput. Achieving high areal densities of physically independent reaction containers (>4/mm2
) requires stringent fluidic isolation between adjacent containers and a high degree of environmental control to prevent cross-contamination and evaporative loss during temperature cycling. Despite these challenges, parallel micro- or nanofluidics offers throughput advantages by thermal cycling and imaging many reactions at once to quantify target copy number in multiple genes and samples, simultaneously. Imaging reactions in parallel allows for longer integration times, improves detected signal-to-noise ratios and benefits PCR specificity and sensitivity by requiring fewer temperature cycles to detect a given target copy number. Shorter cycle times are facilitated by rapid heat transfer across proportionally larger surface areas as the reaction volume is reduced.
Intuitively, an approach to high-throughput qPCR grounded in a high-density array of nanoliter reactions is attractive because it combines the high precision, accuracy and dynamic range of qPCR with the parallelism of a microarray for simultaneous quantification of gene expression across multiple genes and samples. For this to occur, two challenges need to be overcome. The first is creation of a simple interface for precise and accurate transfer of liquids between the wells of a microplate to those of a nanoplate. The second is achieving the accuracy, precision and sensitivity demanded by qPCR in a 96- or 384-well microplate but in a substantially reduced reaction volume. Discovery of a facile interface for speedy transfer of liquids between micro- and nanoplates and identifying a robust approach to ensure qPCR assay performance at the nanoliter-scale has been at the leading edge of our development efforts.
We have solved these problems with an approach based on through-hole arrays (25
). Effectively thought of as a high-density version of a microplate, our nanoplates combine the high-throughput and reagent savings of a nanofluidic system with the macroscale performance of qPCR in microplates. A stainless steel (317 stainless steel) platen the size of a microscope slide (25 mm × 75 mm × 0.3 mm) is photolithographically patterned and etched to form a rectilinear array of 3072, 320 μm diameter through-holes. The through-holes are grouped in 48 subarrays of 64 holes each and spaced on a 4.5 mm pitch equal to that of wells in a 384-well microplate (). A series of vapor and liquid deposition steps covalently attaches a PCR compatible polyethylene glycol (PEG) hydrophilic layer amine-coupled to the interior surface of each through-hole, and a hydrophobic fluoroalkyl layer to the exterior surface of the platen. The differential hydrophilic–hydrophobic coating facilitates precise loading and isolated retention of fluid in each channel. Primer pairs stored in 384-well microplates are transferred into individual through-holes by an array of 48 slotted pins manipulated by a 4-axis robot (XYZθ
) in an environmentally controlled chamber to prevent evaporative loss during loading. Once a platen is fully populated with primer pairs, the solvent is evaporated in a controlled manner leaving the primers immobilized in a PEG matrix on the inside surface of each through-hole. The array loaded with primer is stored in an evacuated Mylar™ bag at −20°C, ready for sample addition.
Figure 1 A stainless steel platen (317 stainless steel) the size of a microscope slide (25 mm × 75 mm × 0.3 mm) is photolithographically patterned and wet etched to form a rectilinear array of 3072 micro-machined, 320 μm diameter holes (more ...)
Up to 48 different, previously prepared cDNA samples at a concentration of 32 ng/μl are mixed with off-the-shelf qPCR reagents for SYBR Green PCR (see Materials and Methods; PCR Mix) and dispensed into each sub-array (one sample per sub-array) with an automated 48 pipette tip dispensing device. A slotted cassette for holding the platen is assembled by sandwiching a U-shaped glass-reinforced epoxy polymer spacer between two microscope slides patterned with an opaque ink to optically mask background autofluorescence from the spacer. A degassed, immiscible perfluorinated liquid (Fluorinert™) is dispensed into the cassette, the platen inserted and the assembly hermetically sealed with a plug of ultraviolet (UV) curable epoxy.
Real time PCR (RT-PCR) occurs in a computer-controlled imaging thermal cycler whose essential components are two pairs of off-axis, high energy light emitting diode (LED) excitation sources, a thermoelectric flat block holding up to three encased arrays, two emission filters in a computer-controlled filter wheel and a thermoelectrically-cooled CCD camera. Under software control, the real time method for 9216 PCR amplifications and dissociation curves is implemented in <4 h. Post-acquisition data processing generates fluorescence amplification and melt curves for each through-hole in the array, from which cycle threshold (CT) and melt temperature (Tm) are computed. All data are stored in a flat file (*.csv) format for ready export to a database or third party software for further analysis.