Microfluidic Nucleic Acid Technologies Alice Macdonald September 28, 2007

Motivation: - want to purify DNA, RNA from cells. - here we can do this in an automated fashion for small numbers of cells and small volumes

Background Lab on a chip Want to analyze single cells Bigger isn’t always better New possibilities for biology Want to analyze single cells Can’t use traditional isolation techniques Fabrication and automation techniques need to be sophisticated to achieve complicated processes

mRNA purification chip Figure (1) mRNA purification chip.(a) Layout of the microfluidic chip. The blue lines represent the 100-m wide fluidic channels, the red lines are the 100-m wide valve actuation channels. The fluidic ports are named; the actuation ports are numbered 1 to 11. The 'lysing buffer chamber' is composed of the channel space delineated by valves 1, 2, 3 and 4. The 'cell chamber' is composed of the channel space delineated by valves 4, 5, 6 and 7 (see also b,c). The 'bead chamber' is composed of the channel space delineated by valves 7, 8, 9 and 10 (see also b). (b) Photograph of the in situ affinity column construction. A column of 2.8-m diameter paramagnetic beads covered with oligo dT is being built against a partially closed microfluidic valve (valve 8, see Fig. 1a). Scale bar, 200 m. (c) One cell is loaded in the 'cell chamber' before the lysis step. (The channels are 100 m wide). Scale bar, 100 m.

Valves and pumps Steve Quake’s genius! Makes this sort of automation possible

DNA purification chip Figure(3)DNA purification chip and schematic diagram of one instance of the DNA isolation process.(Open valve, rectangle; closed valve, x in rectangle.) (a) Bacterial cell culture (red) is introduced into the microfluidic chip through the 'cell in' port located in the uppermost part of the chip. Buffer (green) for dilution of the cell sample is introduced through the 'buffer in' port located next to the 'cell in' port. The amount of dilution is determined by the ratio of channel lengths for cells and buffer; in this case, it is 1:1. Lysis buffer (yellow) is introduced from the left side of the chip. (b) The cell sample, dilution buffer and lysis buffer slugs are introduced into the rotary mixer. (c) The three different liquids are mixed thoroughly and consequently bacterial cells are lysed completely. Mixing is limited by diffusion, and without this rotary mixer complete mixing requires several hours to accomplish. For this reason, three different valves are operated sequentially to circulate the fluid inside the reactor, completely mixing the solutions after several minutes. (d) The lysate is flushed over a DNA affinity column and drained to the 'waste port.' (e) Purified DNA is recovered from the chip by introducing elution buffer from the left side of the chip and can be used for further analysis or manipulation. (f) Integrated bioprocessor chip with parallel architecture. This chip is made of two different layers, the actuation layer and the fluidic layer, which are bonded together. The actuation channels are filled with a green food coloring and the fluid channels are filled with yellow, blue and red food coloring according to their functionalities. The width of the fluid channels is 100 m and that of valve actuation channels is 200 m. Input and output ports and vias have been annotated. Multiple parallel processes of DNA recovery from living bacterial cells are possible in three processors with sample volumes of 1.6 nl, 1.0 nl and 0.4 nl. The chip has 26 access holes, 1 waste hole and 54 valves within 20 20 mm. The control channels are connected to polyethylene tubing through stainless steel pins.

Main Results Main result is showing that this complexity can be achieved - proof of concept Show that the RNA and DNA processes work on the chip

RT-PCR analysis of isolated mRNA Figure (2) RT-PCR analysis of isolated mRNA.(a) A series of experiments, together with negative controls, were done to demonstrate single-cell sensitivity. The RT-PCR products were analyzed on a 2% agarose gel loaded with 5% of the reaction; the amplified gene is -actin. Lane 1, 1 kb ladder; lane 2, PBS, no cells (on the chip); lane 3, one cell (on the chip); lane 4, nine cells (on the chip); lane 5, 200 l supernatant from 1-d-old cells + 5 l of beads (in a test tube); lane 6, 200 l lysing buffer + 5 l of beads (in a test tube); lane 7, cells loaded on the chip but none trapped in the chamber (on the chip); lane 8, 200 l deionized water + 5 l beads (in a test tube); lane 9, PCR reagents only. (b) A second series of experiments were done to test the chips with variable numbers of cells and both -actin and OZF transcripts. The RT-PCR products for both transcripts were analyzed on a 2% agarose gel, whose bands were quantified, normalized and plotted on a graph. Zero values indicate the absence of a detectable band in the gel; neither transcript was detected in the experiment with 19 cells, possibly because of RNAase contamination or chip failure. For high-abundance actin mRNA (closed circles), detection is down to the single-cell level; for the moderately abundant OZF mRNA (open circles), detection is at the level of 2−10 cells.

Recovery of E.coli genomic DNA Figureﾊ4.ﾊVerification of successful recovery of E. coli genomic DNA.The eluted samples were removed from the chip and amplified with PCR. The amplicons were run on a 2.0% agarose gel in 0.5 Tris-Borate-EDTA buffer. The target DNA is a 461-bp fragment of the E. coli gene ppdD. (a) Isolation of genomic DNA with an undiluted E. coli culture. Gel lanes as follows: M, PCR marker; lane 1, isolation of genomic DNA from 1.6 nl of culture with a corresponding bacterial cell number of 1,120; lane 2, a 1.0 nl sample, cell number 700; lane 3, a 0.4 nl sample, cell number 280; lane 4, 5, and 6, negative controls for genomic DNA isolation. Instead of bacterial cell culture sample, purified water was used and all the other conditions were the same as lanes 1, 2, and 3. (b) Isolation of genomic DNA from a 1:10 dilution of bacterial cell culture. Lanes 1, 2, 3, lanes 4, 5, 6, and lanes 7, 8, 9 are the results from three different chips, respectively. Gel lanes as follows: M, PCR marker; lanes 1, 4, and 7, isolation of genomic DNA from an average of 112 bacterial cells; lanes 2, 5, and 8, isolation of genomic DNA from an average of 70 bacterial cells; lanes 3, 6 and 9, isolation of genomic DNA from an average of 28 bacterial cells. (c) Graph shows quantification of gel bands from a and b as a function of the number of bacteria trapped.

Significance Effective large architecture Can analyze as little as a single cell Could replace current macro-scale technologies?

References Wook Hong J, Studer V, Hang G, Anderson WF, Quake SR. A nanoliter-scale nucleic acid processor with parallel architecture. Nature Biotech. 2004; 22:435-9 Melin J, Quake SR. Microfluidic large-scale integration: the evolution of a set of design rules for biological automation. Annu Rev Biophys Bimol Struct. 2007; 36:213-31

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