Printing chemical libraries on microarrays for fluid phase nanoliter reactions

Abstract

Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots/cm2. Using aerosol deposition, subsequent reagents and water were metered into each reaction center to rapidly assemble diverse multicomponent reactions without crosscontamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease-substrate interactions, and high-throughput screening reactions. An inhibitor of caspases 2, 4, and 6 was identified by using a 352-compound combinatorial library microarrayed in quadruplicates on 100 slides and screened against caspases 2, 4, and 6, as well as thrombin and chymotrypsin. From one printing run that consumes <1 nanomole of each compound, large combinatorial libraries can be subjected to numerous separation-free homogeneous assays at volumes 103-104 smaller than current high-throughput methods.

Fig. 1.

(A) Nanoliter volumes of glycerol were contact printed in a 384-microarray format at 400 spots per cm².(B) An aerosol of biological sample generated via an ultrasonic nozzle allowed metering of picoliter to nanoliter quantities of reactants into each individual glycerol reaction center. (C) The array, imaged by differential interference microscopy, is shown before and immediately after aerosol deposition. The deposited aerosol rapidly dried within 7 s leaving the spots intact and maintaining individual reaction compartments. (Bar = 200 μm.) (D) To demonstrate the uniformity of the aerosol deposition process and the absence of spot-to-spot intermixing, an aerosol of TRITC (1 mM in DMSO; yellow) was deposited at 400 nl/s for 4 s on an array initially containing alternating rows of AMC (1 mM; blue) and dye-free glycerol spots. (Bar = 500 μm.)

Fig. 2.

(A) Protease profiling used a 16 × 24 microarray consisting of three fluorogenic substrates and substrate-free spots activated with thrombin (1 unit/ml) and chymotrypsin (200 μM) delivered at a liquid flow rate of 400 nl/s for 4 s. The blue fluorescence resulted from the cleavage of the thrombin substrate (boc-VPR-MCA, 500 μM) and the red (Texas red) and green (fluorescein) fluorescence resulted from the cleavage of the BODIPY TR-X and FL casein substrates (50 μg/ml), respectively, by chymotrypsin. Positions where no color was visible consisted of substrate-free spots. (B) Epifluorescence microscopy with low-level transmitted white light of the highlighted region in A revealed the dried aerosol between reaction centers and complete compartmentalization of each reaction center. (C) In a demonstration of protease-substrate profiling in the presence of inhibitors, six different fluorogenic MCA peptide substrates (250 μM) in the absence (left) or presence of various aldehyde (CHO) peptide inhibitors (500 μM) were microarrayed and then assayed with 1 unit/ml caspase 2 delivered at a flowrate of 400 nl/s for 4 s.

Fig. 3.

(A) Microarrays activated with human thrombin and boc-VPR-MCA displayed a dose-dependent inhibition by the thrombin inhibitor benzamidine (B) as seen in the boxed central zone that contained duplicate reaction centers with increasing concentration of the inhibitor from 0 to 100 mM. All other reaction centers lacked inhibitor allowing full fluorescence generation (coefficient of variation <6%, n = 244). Thrombin (10 units/ml) was delivered at 400 nl/s for 4s, followed by delivery of the fluorogenic substrate, boc-VPR-MCA (10 mM) at 400 nl/s for 4 s. (C) A dose-dependent increase in fluorescent intensity of the reaction was observed. (D) The detection limit for human plasmin cleavage of (CBZ-FR)₂-R110 was 0.1 nM (P < 0.001) in the liquid sample before aerosol deposition.

Fig. 4.

(A) Nanoliter screening of a compound library microarrayed at 1 mM in quadruplicate with human caspase 6, and a fluorogenic substrate (VEID-MCA) identified the location of an inhibitor (compound 1,C₁₆H₁₁Br₃N₄O). When replicate microarrays were screened against caspase 2 (B) or 4 (C), the identical location on the microarrays as seen in A displayed low-fluorescence emission indicative of enzyme inhibition. When tested in triplicate in a standard well plate assay (2 units/μl caspase in 15-μl reaction with 200 μM substrate in buffered saline lacking glycerol and with <0.4% DMSO at 22°C and incubated 1,200 s), compound 1 caused a dose-dependent inhibition of caspases 2, 4, and 6 with an IC₅₀ of ≈0.5–5 mM against the three caspases (D) with 100% inhibition detected in all reactions at 10 mM. Compound 1 had no fluorescence emission or excitation overlap with the assay components.