Digitally programmable microfluidic automaton for multiscale combinatorial mixing and sample processing

Abstract

A digitally programmable microfluidic Automaton consisting of a 2-dimensional array of pneumatically actuated microvalves is programmed to perform new multiscale mixing and sample processing operations. Large (μL-scale) volume processing operations are enabled by precise metering of multiple reagents within individual nL-scale valves followed by serial repetitive transfer to programmed locations in the array. A novel process exploiting new combining valve concepts is developed for continuous rapid and complete mixing of reagents in less than 800 ms. Mixing, transfer, storage, and rinsing operations are implemented combinatorially to achieve complex assay automation protocols. The practical utility of this technology is demonstrated by performing automated serial dilution for quantitative analysis as well as the first demonstration of on-chip fluorescent derivatization of biomarker targets (carboxylic acids) for microchip capillary electrophoresis on the Mars Organic Analyzer. A language is developed to describe how unit operations are combined to form a microfluidic program. Finally, this technology is used to develop a novel microfluidic 6-sample processor for combinatorial mixing of large sets (>2(6) unique combinations) of reagents. The digitally programmable microfluidic Automaton is a versatile programmable sample processor for a wide range of process volumes, for multiple samples, and for different types of analyses.

Fig. 1

(A) Cross sectional view of a microvalve array showing the steps for transfer of fluids between microvalves. (B) Schematic of a combining operation showing labeling of valve array and inputs. Inputs from wells c1 and d2 are drawn in via valves C1 and D2, combined in valve C2, and transferred to output well b1, with a total fluidic transfer of one valve volume per cycle. The cycle is then repeated 100 times. (C) Fluidic program language. Combining operations indicate inputs (x,y…) followed by the microvalve or microvalves where combining occurs (z). Transfer operations indicate the microvalve or storage well input (x) followed by the output (y), and the number of microvalve volumes transferred per cycle (i). Operations within brackets are repeated n times. Flutter operations are performed continuously and indicate the microvalve fluttered (x).

Fig. 2

Six sample combinatorial processor. Fluidic inputs 1–6 can be precisely transferred to the combining valve and digitally transferred through a series of valves to an outlet for storage. Check valves at each fluidic input enable combinatorial selection of any subset of the reagents in a given program cycle, and prevent cross contamination of the reagents. Using this system, 2ⁿ multiscale reagent combinations are enabled by n + 4 microvalves. The selected reagent sets can be represented as a binary code. For instance, selection of inlets 1, 3, and 6 is denoted by the binary string, 101001.

Fig. 3

Epifluorescence images of loaded combining valves and schematics of multiscale sample processing operations on the digitally programmable microfluidic Automaton. (A) Combining valve C3 is loaded with of two fluorescent dyes from parallel input channels. The contents of C3 are then digitally transferred between B3-A1 to a1 in this example. (B) Combining valves loaded with two reagents from orthogonal inputs, and (C) with fluorescent dyes from three different inputs. The contents of a combining valve can be transferred to any microvalve or output in the array.

Fig. 4

(A) Illustration of the program for continuous digital mixing of two reagents. ROX and fluorescein dyes are drawn into combining valve C3 and then digitally transferred through valves B3-A1 to an output well. (B) Epifluorescence images of each step of two different mixing programs. Program 1 includes continuous fluttering (repeated opening and closing of A3 with 50 ms cycles) throughout the program, while Program 2 is run without fluttering. (C) Fluorescence line profiles obtained across valve A1 intersecting the direction of flow during the performance of Program 1 with fluorescein and pure buffer inputs. The vertical lines indicate the location of the first moment of the fluorescence distribution; a sample mixed off chip results in a center at 49 ± 1% of the microvalve width. With a 200 ms valve actuation time, complete mixing occurs within 800 ms during transfer from B3 to A1.

Fig. 5

Multiscale serial dilution program implemented on the Automaton. (A) The first dilution is achieved by combining sample and buffer in two microvalves (dashed box). The contents of two microvalves are then transferred to Out1 at each cycle, resulting in faster generation of large volumes. Each subsequent standard concentration level (B–D) is generated by combining sample from the previous standard curve level and buffer in the two indicated microvalves. (E) Results of the standard curve program generating 6 µL of each concentration level within a total runtime of 7 min. Error is estimated based on four separate runs. An R² of 0.996 is achieved with a predicted dilution factor of 2 for each level.

Fig. 6

(A) Schematic of the program for labeling carboxylic acids performed on the Automaton. Sample containing a standard set of carboxylic acids is loaded into a combining microvalve with Cascade Blue (CB) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (B). At each cycle, the contents of the combining valve are transferred to Out1 with an output rate of 0.06 µL s⁻¹. (C). After a 15 min incubation, the contents of Out1 are combined with 30 mM borate buffer, pH 9.5 for dilution and transferred to Out2. Approximately 30 µL of labeled sample is generated for analysis by microcapillary electrophoresis on the Mars Organic Analyzer or MOA (D). The standard contains 200 µM formic acid, 400 µM each acetic, propanoic, butanoic, pentanoic, hexanoic, and heptanoic acids, and 600 µM octanoic acid.

Fig. 7

Bright field images of all possible reagent subsets loaded into the combining valve of the 6-bit combinatorial mixing device. Green, blue, clear, red, yellow, and black dyes were loaded into reservoirs 1,2,3,4,5, and 6, respectively.