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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Micro Total Anal Syst. Author manuscript; available in PMC 2010 April 21.
Published in final edited form as:
Micro Total Anal Syst. 2008 October 12; 2008: 1904–1906.
PMCID: PMC2857516



We report the construction and testing of a combinatorial multicomponent plug mixer (CMPM) chip that generates a large number of mix ratios. The CMPM chip has been designed to study ribonucleotide reductase (RNR) protein-protein/protein-ligand interaction networks. The 4-component chip is capable of 5400 different combinations in a 30 plug cycle. CMPM chips were tested producing fluorescent dye and dihydrofolate reductase NADPH/MX mixtures with plug lengths of 2 mm.

Keywords: Chemical signals, CMPM, PDMS, RNR


Systems chemistry deals with the emergent properties of interacting chemical systems or networks [1]. These properties result from the interaction between the components in a complex network. The study of such phenomena requires the generation of a large number of mix ratios in an efficient manner with minimal sample consumption. In this paper we report a combinatorial multicomponent plug mixer (CMPM) microfluidic chip which is capable of mixing four different components in a wide range of ratios. The CMPM chip has been designed to study complex ribonucleotide reductase (RNR) protein-protein/protein-ligand interaction networks reconstituted in vitro. The RNR system is critical to dNTP/DNA production and agents used clinically to treat cancer (e.g. hydroxyurea and gemcitabine).


Figure 1(a) shows a block diagram of the multicomponent mixer chip. The CMPM accepts a vector of M reactive reagents (C1,C2,C3, …, CM) input flows and generates a continuous flow exit consisting of a stream of analyte plugs which rapidly mix by dispersion (i.e. sequential segmentation [2]) followed by chemical equilibrium. The equilibrium mixture is next analyzed by either fluorescence analysis or mass spectrometry. The initial constitution of the mixture is set by repeating N-combinations of short analyte plugs carried by a buffer flow. Each plug has equal time duration TP. Figure 1(b) shows an illustrative example of the generation of (C1:2C2:C3:C4)/N mixture. For a mixture stream with N plugs and M reagents (inclusive of buffer), the number of possible mix combinations is C(N1,N)=(N1)!/N!/(M-1)! Periodic plug streams with 20 plugs can thus generate billions of combinations with this simple scheme. Figure 2 shows a schematic of a four-reagent CMPM. The plug output flow is directed to a long capillary/storage region where the mixture can be incubated. Figure 3 shows a photograph of the chip implemented using two-level PDMS technology [3] with through-level vias [2]. The channel dimensions were 16×125 µm2. The chip measures 1.5×1.8 cm2.

Figure 1
(a) The CMPM accepts a vector of multiple analyte flows and generates repeating streams of analyte plugs that control the mixture composition. (b) Illustration of a (C1:2C2:C3:C4)/N mixture formation.
Figure 2
Schematic of four reagent CMPM. The multiplexer routes a series of discrete plugs to the output flow resistor that averages the plugs yielding the desired output mixture concentration.
Figure 3
Four-reagent CMPM chip implemented with two-level PDMS technology. The chip dimensions are 1.5×1.8 cm2.


The chip was tested using three water soluble fluorescent dyes: clear blue (IFWB-C0), fluorescent red (IFWB-C7) and fluorescent yellow-green (IFWB-C8) from Risk Reactor (Ca) mixed in H2O at 50:1000, 1:1000 and 1:1000 ratios, respectively. The concentration of the output dye mixture was measured using an Olympus MVX10 fluorescence imaging microscope. Figure 4 shows photographs of the chip in operation loading dye plugs into the exit channel at TP= 100 ms at average flow velocity of 2.0 cm/s, with 2 mm unit plug length. Figure 5 shows results a few measured exit concentration scans for a 10 plug, 20 cycle example with 64 different dye combinations recorded ~6 cm downstream from the multiplexers. Figure 6 shows a three dimensional scatter plot of the recorded dye mixtures. The decrease in the amplitude for C2 was the result of dye interaction with C1.

Figure 4
Fluorescence images of the three valves injecting plugs. The plug duration was 100 ms.
Figure 5
Composition of exit mixture for each dye averaged by the output flow resistor at ~6 cm downstream from the flow multiplexers. The fluorescence data was recorded at 2.5 images/s representing a 64-level sweep.
Figure 6
Three-dimensional scatter plot of the dye mixture combinations generated by the CMPM. Many more combinations can be generated with a larger number of plugs N.

The chip was next used to perform initial experiments of the titration of dihydrofolate reductase with (fluorescent) NADPH [4] (nicotinamide adenine dinucleotide phosphate) in a stopped-flow fluorescence configuration. Dihydrofolate reductase catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate(THF). The enzyme is essential for thymidylate biosynthesis and hence for DNA synthesis. Figure 7 shows the recorded NADPH fluorescence traces using a DAPI blue filter and Olympus MVX 10 microscope.

Figure 7
Titration of dihydrofolate reductase, MTX/R and NADPH [4] at different NADPH: protein-enzyme ratios. The protein solution was made by mixing 1 ml reaction buffer, 1.4 µl S-DHFR and 1 µl of MTX. The base (corresponding to 1/20) volume and ...


We demonstrated a microfluidic combinatorial mixer chip for systems chemistry studies capable of generating thousands (4 inputs, 30 plugs) to billions (20 inputs, 20 plugs) of mix ratios.


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