Langmuir 2000, 16, 9709-9715
9709
Encoding Combinatorial Libraries: A Novel Application of Fluorescent Silica Colloids Lisbeth Grøndahl,† Bronwyn J. Battersby,† Darryn Bryant,‡ and Matt Trau*,† Department of Chemistry and Department of Mathematics, The University of Queensland, St. Lucia QLD 4072, Australia Received July 13, 2000. In Final Form: September 8, 2000 A major challenge associated with using large chemical libraries synthesized on microscopic solid support beads is the rapid discrimination of individual compounds in these libraries. This challenge can be overcome by encoding the beads with 1 µm silica colloidal particles (“reporters”) that contain specific and identifiable combinations of fluorescent dyes. The colored bar code generated on support beads during combinatorial library synthesis can be easily, rapidly, and inexpensively decoded through the use of fluorescence microscopy. All reporters are precoated with polyelectrolytes [poly(acrylic acid), PAA, poly(sodium 4-styrenesulfonate), PSSS, polyethylenimine, PEI, and/or poly(diallyldimethylammonium chloride), PDADMAC] with the aim of enhancing surface charge, promoting electrostatic attraction to the bead, and facilitating polymer bridging between the bead and reporter for permanent adhesion. As shown in this article, reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to quantity of attached reporters per bead (54 ( 23 in 2500 µm2 area for PEI/PAA coated and 11 ( 6 for uncoated reporters) and minimization of cross-contamination (1 red reporter in 2500 µm2 area of green-labeled bead for PEI/PAA coated and 26 ( 15 red reporters on green-labeled beads for uncoated reporters after 10 days). Examination of various polyelectrolyte systems shows that the magnitude of the ξ-potential of polyelectrolyte-coated reporters (-64 mV for PDADMAC/PSSS and -42 mV for PEI/PAA-coated reporters) has no correlation with the number of reporters that adhere to the solid support beads (21 ( 16 in 2500 µm2 area for PDADMAC/PSSS and 54 ( 23 for PEI/PAA-coated reporters). The contribution of polymer bridging to the adhesion has a far greater influence than electrostatic attraction and is demonstrated by modification of the polyelectrolyte multilayers using γ irradiation of precoated reporters either in aqueous solution or in polyelectrolyte solution.
Introduction Large chemical libraries play a vital role in technologies such as drug discovery and gene screening.1 These libraries may consist of a mixture of chemical species in solution2 or may be synthesized on polymer or glass beads (2-500 µm) via the combinatorial split-and-mix method.3 The latter method of compound synthesis can efficiently produce libraries containing billions of compounds, with each support bead possessing only one compound. In fact, the number of “discrete” compounds in these libraries is limited only by the volume of beads that can be practically handled. One major challenge associated with using large libraries produced by the split-and-mix procedure is rapid and simple identification of individual compounds on the support bead. Direct analysis of any chosen compound from these libraries is generally prevented due to the small quantities available from one bead. The conventional method of chemically labeling the beads with “identifier tags” (e.g. oligonucleotides, secondary amines, fluorophenyl ethers, fluorescent molecules) involves extra chemical reactions to covalently attach the tags. The necessity for compatible compound and tag synthesis reactions places limitations on the procedure.4 We recently introduced an * Corresponding author: Telephone: +61 7 3365 3816. Fax: +61 7 3365 4299. E-mail:
[email protected]. † Department of Chemistry. ‡ Department of Mathematics. (1) (a) Lee, M. S.; Nakanishi, H.; Kahn, M. Curr. Opin. Drug Discovery Dev. 1999, 2, 332. (b) Fox, S.; Farr-Jones, S.; Yund, M. A. J. Biomol. Screening 1999, 4, 183. (2) Quinn, R. J. Drug Dev. Res. 1999, 46, 250. (3) (a) Furka, A Ä .; Sebestye´n, F.; Asgedom, M.; Dibo´, G. Int. J. Pept. Protein Res. 1991, 37, 487. (b) Lam, K. S.; Lebl, M.; Krchna´k, V. Chem. Rev. 1997, 97, 411.
alternative method, “colloidal bar coding”, which involves encoding the support beads during split-and-mix syntheses with silica colloids (1-3 µm “reporters”) that contain specific and identifiable combinations of fluorescent dyes.5 Using this technique, it is possible to encode a library of more than 16 million compounds with just six fluorescent dyes.5 Six dyes can be synthesized into reporter particles in 64 different combinations, and each type of reporter can be used to encode one reaction in the split-and-mix synthesis. The concept of colloidal bar coding was described in detail by Battersby et al.5 and is briefly outlined below. In our split-and-mix process, the solid support beads are first split into several portions and each portion is mixed with a different reporter suspension. The reporter suspensions are distinguishable by the reporters they contain; reporters within a suspension are identical (i.e., same dye combination and size), but each suspension contains a different type of reporter. The support beads in each portion become coated with multiple reporters (>50 reporters per bead) of the same type (Figure 1). Each portion is then reacted with a different monomer (e.g. amino acid, nucleic acid, etc.), and the portions are recombined to complete a cycle (Figure 2). The above processes are repeated for a chosen number of cycles, resulting in an encoded chemical library ideally consisting of all monomer and reporter combinations. The various (4) (a) Ni, Z.-J.; MacLean, D.; Holmes, C. P.; Murphy, M. M.; Ruhland, B.; Jacobs, J. W.; Gordon, E. M.; Gallop, M. A. J. Med. Chem. 1996, 39, 1601. (b) Czarnik, A. W. Curr. Opin. Chem. Biol. 1997, 1, 60. (c) Egner, B. J.; Rana, S.; Smith, H.; Bouloc, N.; Frey, J. G.; Brockelsby, W. S.; Bradley, M. Chem. Commun. 1997, 735. (d) Scott, R. H.; Balasubramanian, S. Bioorg. Med. Chem. Lett. 1997, 7, 1567. (e) Campian, E.; Sebestye´n, F.; Major, F.; Furka, A. Drug Dev. Res. 1994, 33, 98. (5) Battersby, B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau, M. J. Am. Chem. Soc. 2000, 122, 2138.
10.1021/la000995z CCC: $19.00 © 2000 American Chemical Society Published on Web 11/10/2000
9710
Langmuir, Vol. 16, No. 25, 2000
Grøndahl et al.
Figure 1. Scanning electron microscope image of the surface of a solid support bead encoded with multiple colloidal silica particles 2.5 µm in diameter.
fluorescent bar codes that are generated on the support beads during library synthesis are a record of the reaction history of each bead. Each reporter encodes for a particular monomer in a particular cycle in the split-and-mix synthesis. Reading the bar code, by imaging the bead through various filters in a fluorescence microscope, permits accurate and inexpensive decoding of the combinatorial sequence generated on the bead during compound synthesis. Thus, the compound attached to the bead is unambiguously identified. Described herein are the methods used to obtain robust adhesion of the silica colloids to solid support beads. As can be appreciated, robust adhesion of the small reporters to the solid support beads is absolutely critical for the viability of this method. This adhesion is achieved through the use of controlled polyelectrolyte (charged polymer) multilayer coating of reporters. Encapsulation of particles with polymer multilayers has been previously reported for applications in the medical, pharmaceutical, agricultural, and cosmetic industries for the development of controlled-release delivery systems.6 The new application for polymer multilayer-coated particles that is presented here involves coating all reporters with polyelectrolytes, prior to library synthesis. This coating is designed to enhance surface charge, promote electrostatic attraction to the bead, and facilitate polymer bridging between the bead and reporter for permanent adhesion. Factors that influence the robustness of the colloidal bar code and number of reporters that adhere to each support bead under various conditions were investigated and are discussed herein. These factors include reporter concentration, contact time of reporter solution with beads, reporter surface charge, and polyelectrolyte multilayer assembly. Ionizing radiation was used as a method of modifying the molecular structure of the adsorbed polyelectrolyte layer, and data that indicate that reporter adhesion is driven more by polymer bridging flocculation than by attractive electrostatic forces are presented. (6) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. Langer, R. Nature 1998, 392, 5.
Figure 2. Use of colloidal bar coding in a split-and-mix synthesis. (A) Support beads are split into a chosen number of equal portions. (B) Fluorescent silica “reporters” are attached to each bead. Each portion of support beads is encoded with a unique type of reporter that contains a distinct combination of fluorescent dyes.5 (C) A different monomer (e.g. an amino acid) is reacted with each portion. (D) The encoded beads are recombined to complete the cycle.
Materials and Methods Materials. Monodisperse silica particles, functionalized with carboxylic acid (-COOH) groups, were purchased from Micromod (Germany) and used as reporters. Four types of reporter were used, each type containing one of the following fluorescent dyes or dye combinations: Rhodamine (red-emission; 900 nm mean diameter), aminofluorescein (green emission; 700 nm mean diameter), DAPI (blue emission; 1 µm mean diameter), and aminofluorescein/Rhodamine (red/yellow emission; 1 µm mean diameter). The solid support beads were acryloylated O,O′-bis(2-aminopropyl)poly(ethylene glycol)/acrylamide copolymer resin (PL-PEGA; 0.4 mmol/g substitution; 150-300 µm diameter) and were obtained from Polymer Laboratories. The polyelectrolyte, poly(acrylic acid) (PAA; Mn ) 250 000), was purchased from
Encoding Combinatorial Libraries Scientific Polymers. Other polyelectrolytes, poly(sodium 4-styrenesulfonate) (PSSS; Mn ) 1 000 000), polyethylenimine (PEI; Mn ) 10 000), and poly(diallyldimethylammonium chloride) (PDADMAC; Mn ) 400 000-500 000), were obtained from Aldrich. Milli-Q water was used throughout, and all solvents were of analytical grade. Polyelectrolyte Coating of Reporters. Positively charged polyelectrolytes (PEI or PDADMAC) were adsorbed onto the silica reporters by mixing 3 mL of 1% polyelectrolyte solution in water with 10 mg of dry reporters, sonicating for 30 min, equilibrating for 24 h, and then washing thoroughly six times with 3 mL of water. The second, negative coating was adsorbed to the positively charged reporters by mixing 3 mL of 1% solution of negative polyelectrolyte (PAA or PSSS) with the positively coated reporters suspended in small amounts of water, leaving it for 24 h, and washing as above.7 An aliquot of 2 mL of the aqueous suspension of silica reporters was centrifuged, the supernatant removed, and the silica reporters washed three times with 3 mL of DMF and stored finally in 2 mL of DMF, yielding a reporter concentration in DMF of 3.3 g/L. Radiation Grafting of Polyelectrolyte-Coated Reporters. Silica particles coated with polyelectrolytes were γ-irradiated in the presence or absence of negatively charged polyelectrolyte (PAA or PSSS). Radiation grafting was performed with a Gammacell 220 using a Cobalt-60 source with a dose rate of approximately 7.6 kGy/h. When aqueous suspensions of polyelectrolyte-coated silica reporters were exposed to γ-irradiation, a dose of 1.3-3.8 kGy was employed. Alternatively, when polyelectrolyte-coated silica reporters were exposed to γ-irradiation in 0.5% polyelectrolyte suspensions, a dose of 12.4 kGy was employed. ξ-Potential Measurements.. The ξ-potential was calculated using the Smoluchowski relation,8 ξ ) uη/, where u, η, and are the electrophoretic mobility, the viscosity, and the permittivity of the solution, respectively. Electrophoretic mobility, u, was measured at room temperature on polyelectrolyte-coated and uncoated samples of 0.03 mg silica reporters in 3 mL of sodium phosphate buffer (1 mM, pH 6.5), on a custom designed glass capillary of 8.5 × 10-2 m with electrodes at each end. A voltage of 112 V was applied, and a 3-CCD color video camera (JVCKY-F55B) was used to monitor the time taken for particles to pass a view of 0.44 × 10-3 m magnified by a microscope. The image was transmitted to a 17 in. monitor. An average of at least 10 measurements was used to calculate the ξ-potential. Attachment of Polyelectrolyte-Coated Silica Reporters to Resin Beads. In general, 10 mg of PEGA solid support beads was swelled in 500 µL of DMF for at least 5 min before adding an aliquot of the reporter-DMF solution, ranging from 0.25 µL to 25 µL. The mixture was shaken for a given time, and a sample was taken for viewing under the fluorescence microscope. In concentration-dependence studies, mixing of the swelled PEGA beads and PDADMAC/PAA-coated reporters in DMF was performed in a plastic vial for 30 s. The solid support beads were allowed to settle, after which the supernatant was removed. The beads were then washed five times with 500 µL of DMF before samples were taken. In contact-time studies, the swelled PEGA beads were mixed with two different amounts of PDADMAC-PAA-coated reporters in DMF (yielding reporter concentrations of 0.026 and 0.065 g/L) in a plastic vial. The first sample was taken after 30 s, and mixing was continued, with further samples taken at appropriate times. In reporter-attachment experiments, reporters with various types of polyelectrolyte coatings in DMF were added to the swelled solid support beads in a plastic vial (reporter concentration ) 0.026 g/L) and the sample was taken after a mixing time of 5 min. The robustness of the colloidal bar code was examined by mixing together green-labeled and red-labeled PEGA beads and measuring the number of contaminant reporters (i.e., red reporters on green-labeled beads and green reporters on redlabeled beads). Cross-contamination and the detachment sta(7) Matthews, D. A Novel Approach to Encoding/Decoding Reaction Sequences in Combinatorial Chemistry. Honours Thesis, Department of Chemistry, The University of Queensland, Australia, 1998. (8) Von Smoluchowski, M. Z. Phys. Chem. 1918, 92, 129.
Langmuir, Vol. 16, No. 25, 2000 9711 tistics of reporters were examined for cases where the reporters were uncoated or coated with polyelectrolytes (PDADMAC/PAA and PEI/PAA). To prepare each suspension of red- or greenlabeled beads, the appropriate reporters in DMF were mixed with swelled PEGA beads (reporter concentration ) 0.026 g/L) for 16 h. Washing with 500 µL of DMF was performed five times by centrifuging the suspensions and removing the supernatant. The suspensions were gently rotated overnight in order to adsorb any reporters not removed by the washing procedure. Each suspension containing labeled solid support beads was washed once more. The suspension, containing beads labeled with red reporters, was mixed with the beads labeled with green reporters. Each mixture was gently rotated, with samples taken at various times over a 10-day period. The number of green and red (contaminant) reporters within a selected focused area of 2500 µm2 of 10 green-labeled beads was counted at each time (see below). The same procedure was followed to count the number of red and green (contaminant) reporters on red-labeled beads. Analysis of Reporter Coverage. The labeled solid support beads were imaged using a SPOT Diagnostic Instruments camera attached to an inverted fluorescence microscope (Olympus IX70). The microscope was equipped with three filters, namely U-MWU (excitation wavelength λEX ) 330-385 nm; emission wavelength λEM > 420 nm), U-MWB (λEX ) 450-480 nm; λEM > 515 nm), and U-MWG (λEX ) 510-550 nm; λEM > 590 nm). The swelled PEGA solid support beads varied in size with diameters ranging from 0.17 to 0.34 mm, and the top or bottom surface of each bead was imaged. The number of reporters within a selected focused area of 2500 µm2 was counted from these images, and the total number of reporters per bead was an average of at least five images of different beads. Micrographs of single solid support beads with attached silica reporters (magnification ) 200×) were analyzed with ImagePro Plus 4.0 Software. An area of interest (AOI) of 50 µm × 50 µm in the center of the image was selected, and silica reporters within the selected AOI, that displayed a chosen fluorescent dye combination, were counted (Figure 3). The total area of all counted objects within the AOI was divided by the area of a single silica reporter to yield the number of silica reporters within the AOI. The area of a single silica reporter was estimated from a selection of fluorescent objects that could unambiguously be assigned to a certain number of reporters. Due to differences in overall brightness of the solid support beads between micrographs, the apparent area of a single silica reporter varied slightly and was readjusted for each bead image. The counted number of silica reporters per 50 µm × 50 µm area obtained from 5-28 different solid support bead images was subjected to the Q-test9 using a confidence level of 95% before the average number of silica reporters together with the standard deviation was calculated.
Results and Discussion The colloidal bar coding technique involves attachment of monodispersed silica particles (1 µm reporters) to solid support beads on which chemical synthesis is performed. The reporters are coated with polyelectrolyte multilayers with the aim of enhancing surface charge, promoting electrostatic attraction between reporters and resin beads, and permitting polymer bridging of adsorbed polyelectrolyte strands from the reporters to the resin beads for increased reporter adhesion. Influence of Reporter Concentration and Contact Time on Reporter Coverage. The influence of reporter concentration and contact time on the number of reporters that adhere to solid support beads was investigated. The number of attached reporters was manipulated by varying the concentration of reporters in contact with the solid support beads (Figure 3). As shown in Table 1, the average number of reporters that remain adhered to a solid support bead increases substantially with increasing reporter concentration. At the lowest concentration investigated, approximately 1000 reporters were present for every bead (9) Dean, R. B.; Dixon, W. J. Anal. Chem. 1951, 23, 636.
9712
Langmuir, Vol. 16, No. 25, 2000
Grøndahl et al. Table 1. Influence of Reporter Concentration on the Number of PDADMAC/PAA-Coated Green Reporters that Attach to a 2500 µm2 Area on PEGA Beads (10 mg in 500 µL of DMF) in 30 sa
a
[reporter] (g/L)
no. of reporters
0.002 0.013 0.026 0.065 0.159
1(1 8(7 9(8 45 ( 21 81 ( 26
All samples were washed five times to remove excess reporters.
Table 2. Dependence of Mixing Time of Reporter Solution with PEGA Beads (10 mg in 500 µL of DMF) on the Number of Reporters within a 2500 µm2 Area on the Beadsa no. of reporters mixing time (min) [reporter] ) 0.065 g/L [reporter] ) 0.026 g/L 0.5 2 10 100 1 day 7 days
21 ( 13 59 ( 36 77 ( 28 81 ( 39 186 ( 90 269 ( 124
9(8 20 ( 12 22 ( 10 42 ( 38 81 ( 48 90 ( 53
a PDADMAC/PAA-coated green reporters (0.065 or 0.026 g/L) were mixed with the beads for the given time.
Figure 3. Influence of reporter concentration on reporter coverage of PEGA support beads. (A) Fluorescence microscope image of a typical bead when a low concentration of silica reporters (0.002 g/L) is used. One reporter is visible within the 2500 µm2 area of interest (AOI, i.e., the white square). (B) When a higher reporter concentration of 0.065 g/L is used, better reporter coverage is observed. The image in this example shows 47 reporters within the 2500 µm2 AOI. Reporters in both images were coated with PDADMAC/PAA multilayers.
in the suspension, but only very few reporters (0-2) were found to be firmly attached to a bead within the focused 2500 µm2 area. This corresponds to approximately 100 reporters attached to the average bead. With an increase in reporter concentration of 100 times, an average of 80 reporters was found within the 2500 µm2 area. This corresponds to approximately 8000 reporters on a bead. Furthermore, changing the contact time between the reporter solution and the solid support beads can vary the number of reporters on each resin bead. With an increase in the contact time, a gradual increase in the number of attached reporters is observed (Table 2). Two different reporter concentrations were studied, and both indicated
a continued increase in the average number of reporters per 2500 µm2 area of support bead. These results indicate that reporter concentration and contact time can be manipulated to attach a chosen number of reporters to the solid support beads. This suggests that the process may be optimized for any given combinatorial synthesis protocol. Contribution of Colloidal Forces to Reporter Adhesion. The coating of colloids with alternating charged polyelectrolyte multilayers is a well-known method for increasing the stability of a colloidal suspension.10 As described in Battersby et al.,5 polyelectrolyte coating of fluorescent colloids can also be used as a method of optically encoding support beads in solid-phase synthesis. In the study described herein, reporters coated with various polyelectrolyte multilayer assemblies were examined and reporter coverage was measured by analysis of the number of reporters within a 2500 µm2 area on a support bead. Reporters coated with alternating charged polyelectrolyte multilayers adhere to the support beads in significantly greater amounts than do uncoated reporters (for the same reporter concentration and contact time). Not only do the polyelectrolyte coatings generally increase the negative surface charge on the reporters (as shown by the more negative ξ-potentials in Table 3), but the different polyelectrolyte multilayer assemblies affect the number of reporters that adhere to the beads. Although the reporters coated with the PDADMAC/PSSS polyelectrolyte assembly have the highest ξ-potential (-64 mV, Table 3), and should have the highest electrostatic attraction to the positively charged PEGA beads, these reporters adhere to the solid support beads in much lower numbers than do reporters displaying the relatively low ξ-potential of -42 mV (i.e., the PEI/PAA system, Table 3). In fact, the magnitude of the ξ-potential of polyelectrolyte-coated reporters appears to have no correlation with the number of reporters that remain attached to the solid support beads. (10) For review see: Decher, G. Science 1997, 277, 1232.
Encoding Combinatorial Libraries
Langmuir, Vol. 16, No. 25, 2000 9713
Table 3. Variation in ξ-Potential (1 mM Phosphate Buffer, pH 6.5) and the Number of Reporters within a 2500 µm2 Area on PEGA Beads (10 mg in 500 µL of DMF) for Reporters (0.026 g/L) Coated with Various Polyelectrolyte Multilayer Combinationsa polyelectrolyte multilayer combination
ξ-potential (mV)
no. of reporters
none PEI/PAA PEI/PSSS PDADMAC/PAA PDADMAC/PSSS
-41 ( 7 -42 ( 11 -51 ( 16 -54 ( 14 -64 ( 15
11 ( 6 54 ( 23 53 ( 45 28 ( 21 21 ( 16
a
The reporter-bead contact time was 5 min.
These results indicate that, although the polyelectrolyte coating possibly enhances the electrostatic attraction between reporter and solid support bead, contribution to the adhesion by polymer bridging seems to be of greater importance in determining the number of reporters that remain attached to the solid support beads. To further investigate this role of polyelectrolytes, the multilayer coatings on the reporters were modified through crosslinking by γ-irradiation of the reporters. The number of γ-modified reporters that attach to the support beads was then investigated and compared to results obtained with the reporters before their irradiation. Ionizing radiation effects on polymers include crosslinking and scission of chains and modification of the molecular structure.11 γ-irradiation can result in molecular weight changes that can markedly influence the material properties. The relationship between the cross-linking (which leads to network formation), the scission (which leads to a decrease in molecular weight), and the molecular structure of the polymer is important and has been well documented.12 In this study, polyelectrolyte-coated silica reporters were irradiated in the presence and absence of negatively charged polyelectrolyte (0.5% PAA or PSSS) with the goal of modifying the polyelectrolyte network on the reporters. It was anticipated that those reporters irradiated in polyelectrolyte solution should possess a thick “hairy” cross-linked mesh on the reporter surface, with tendrils of polyelectrolyte extending from the reporters into solution. The poly(ethylene glycol)/acrylamide (PEGA) beads used as solid supports have an acrylamide resin backbone with pendant PEG chains that possibly could interact strongly with the polyelectrolyte tendrils on the “hairy” reporters, forming a permanent interlocking molecular web between the reporters and the beads. Conversely, in the absence of free polyelectrolyte in solution, irradiation of the established polymer coating on the reporter should result in a compact network of cross-linked polyelectrolyte which would not be expected to adhere as strongly to the PEGA beads. In all cases, where reporters were coated with one of four different polyelectrolyte multilayer assemblies and γ-irradiated in polyelectrolyte solution, the reporters adhered to support beads in greater quantities than those reporters that were not irradiated (Table 4). The reporters became less negatively charged upon irradiation, as shown by the decreases in ξ-potential, but the number of reporters that adhered to the support beads actually increased by up to 50%. Clearly, not only is polymer bridging, rather than electrostatic attraction, playing the most crucial role in adhesion, but also the extent of polymer bridging is (11) Bowmer, T. N.; O’Donnell, J. H.; Winzor, D. J. J. Polym. Sci. 1981, 19, 1167. (12) Chapiro, A. Radiation Chemistry of Polymeric Systems; Interscience: London, 1962.
Table 4. Influence of γ-Irradiation on the ξ-Potential of Polyelectrolyte-Coated Reporters (0.026 g/L) and on the Number of Reporters within a 2500 µm2 Area on PEGA Beads (10 mg in 500 µL of DMF) after Mixing for 5 mina polyelectrolyte multilayer
irradiation solution
dose (kGy)
ξ-potential (mV)
no. of reporters
PEI/PAA
0.5% PAA
PDADMAC/PAA
0.5% PAA
PEI/PSSS
0.5% PSSS
PDADMAC/PSSS
0.5% PSSS
PEI/PAA
H2O
PDADMAC/PAA
H2O
0 12.4 0 12.4 0 12.4 0 12.4 0 1.3 3.8 0 3.8
-42 ( 11 -40 ( 10 -54 ( 14 -35 ( 8 -51 ( 16 -48 ( 11 -64 ( 15 -52 ( 12 -42 ( 11 -33 ( 12 -33 ( 10 -54 ( 14 -28 ( 8
54 ( 23 90 ( 39 28 ( 21 59 ( 34 53 ( 45 71 ( 24 21 ( 16 24 ( 12 54 ( 23 38 ( 24 14 ( 17 28 ( 21 8(7
a Reporters were transferred to sodium phosphate buffer (1 mM; pH 6.4) prior to ξ-potential measurement.
dependent upon the polyelectrolyte network on the reporter surface. Reporters with PAA as the outer layer, and irradiated in PAA, showed a 40-50% improvement in the number of reporters that adhere. These reporters appear to exhibit polymer bridging to a greater extent than reporters do with PSSS as an outer layer (and irradiated in PSSS) where a 12-25% increase in the reporter coverage is observed. The extent to which polymer bridging occurs between reporter and support bead can be further determined by examining reporters that possess a compact polyelectrolyte mesh. Polyelectrolyte-coated silica reporters were irradiated in the absence of free polyelectrolyte in solution with the aim of forming cross-links between the adsorbed polyelectrolytes already on the reporter surface, thereby stabilizing the polyelectrolyte coat as a compact network. This was achieved through a low irradiation dose (1.3 or 3.8 kGy), as shown by the stability of the modified polyelectrolyte coating upon sonication; a process that for unmodified coatings causes the polyelectrolyte layer to desorb. The formation of the compact, cross-linked network on the reporters actually reduced the number of reporters that adhere to the support beads. With increasing γ-irradiation dose, the ξ-potential became less negative, and the number of reporters attached gradually decreased by more than 70% after a dose of only 3.8 kGy. The reduced reporter coverage is likely to be due to the decreased concentration of polyelectrolyte “tendrils” on the reporters. The mobile, polyelectrolyte chains that are present prior to irradiation become substantially cross-linked during formation of the compact network on the reporter surface. Limiting the mobility of the polyelectrolyte chains decreases interaction with the pendant PEG chains on the PEGA beads, leading to a less secure adhesion and a lower number of attached reporters. Thus, an increase in the number of attached reporters is observed for reporters that possess a “hairy” mesh on their surface, and a decrease occurs when reporters that have a compact mesh are used. This indicates that polymer bridging is having a profound influence on the adhesion. These results suggest that electrostatic forces do not contribute to any significant extent to permanent reporter adhesion, although they may contribute to the initial attraction between the reporters and the support beads. There appears to be no correlation between the magnitude of the ξ-potential and the likelihood of abundant reporter attachment to a bead. Further work could be done to optimize the polyelectrolyte coatings for robust adhesion
9714
Langmuir, Vol. 16, No. 25, 2000
Grøndahl et al.
Table 5. Robustness of the Colloidal Bar Codea polyelectrolyte multilayer none PDADMAC/PAA PEI/PAA
no. of green reporters no. of red reporters on red-labeled beads on green-labeled beads 1 h 1 day 10 days 1h 1 day 10 days 2 0 0
3 0 0
6 1 1
11 ( 3 20 ( 11 26 ( 15 0 0 1 0 0 1
a Comparison of the number of contaminant reporters found in a focussed 2500 µm2 area of PEGA solid support beads at various times after mixing red-labeled (10 mg in 500 µL) and green-labeled beads (10 mg in 500 µL). The incidence of contamination when using uncoated reporters was compared to that which occurred when polyelectrolyte-coated reporters were used.
under different conditions, for example, by varying the radiation dose or investigating different polymers and molecular weights. Robustness of Reporter Adhesion. The colloidal bar coding method of encoding combinatorial libraries requires robust reporter adhesion to permit correct identification of compounds attached to the support beads. The colloidal bar code must be an accurate record of the reaction history of a bead, and thus, exchange of reporters from one bead to another should ideally be prevented. A certain level of cross-contamination can be tolerated before decoding becomes inaccurate (or impossible). As shown below, coating the reporters with polyelectrolytes can control the cross-contamination. Over a period of 10 days, mixtures of red-labeled and green-labeled beads were gently rotated and the reporter exchange between the beads was investigated. The amount of contamination that occurred when uncoated reporters were used was compared to the contamination when nonirradiated polyelectrolyte-coated (PDADMAC/PAA and PEI/PAA) reporters were used. Extremely good results were obtained for both polyelectrolyte assemblies (Table 5). No contamination was observed after 1 day, and an average of only one contaminant reporter appeared in the focused area after 10 days. Thus, the level of cross-contamination was extremely low, with only one contaminant reporter present among the 60 ( 20 correct reporters in the focused area. This means that the contaminant reporters could be ignored and all of the red-labeled beads could be distinguished from the green-labeled beads with 100% accuracy. In contrast, cross-contamination when uncoated reporters were used was so high that it became impossible to distinguish the red-labeled from the green-labeled beads (Table 5 and Figure 4). For example, the number of contaminant red reporters on green-labeled beads was often similar to the number of correct red reporters on red-labeled beads, making the beads impossible to distinguish. The amount of red reporter contamination on green-labeled beads was significantly higher than the amount of green contamination on red-labeled beads. This may be due to the difference in size between the green reporters (700 nm) and the red reporters (900 nm), with the smaller green reporters adhering slightly more strongly. These results are further verification that coating reporters in polyelectrolyte greatly enhances the reporter adhesion and stability of the colloidal bar code. Conclusions This study has indicated that colloidal forces such as polymer bridging flocculation can be manipulated in order to obtain permanent reporter-to-bead adhesion. Moreover, these adhesive forces are shown to be sufficiently strong to minimize the cross-contamination of reporter particles
Figure 4. Fluorescence microscope image of cross-contamination on a support bead encoded with green reporters. The bead was randomly selected from a mixture containing beads labeled with uncoated red or uncoated green reporters. On this greenlabeled bead, a high number of contaminant red reporters (19) were found among the 60 correct green reporters within the 2500 µm2 area of interest (i.e., the white square). The uncoated reporters contaminate other beads at a much higher extent than polyelectrolyte-coated reporters.
from one solid support bead to another. These results are important for development of colloidal bar coding as a method of encoding combinatorial chemical libraries and, coupled with earlier results,5 indicate that the reporterto-bead adhesion is likely to survive multiple cycles in split-and-mix syntheses of chemical libraries. The concentration and time dependencies have important implications for combinatorial synthesis of chemical libraries and the suitability of colloidal bar coding for synthesis of libraries of any type. Control of reporter coverage in the early cycles of combinatorial syntheses will ensure that there is sufficient surface area on each bead to attach reporters in the later cycles. Reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to permanent reporter adhesion, quantity of attached reporters per bead, and minimization of cross-contamination. Examination of various polyelectrolyte systems shows that the magnitude of the ξ-potential of polyelectrolyte-coated reporters appears to have no correlation with the number of reporters that remain attached to the solid support beads. The contribution of polymer bridging appears to have a far greater influence than electrostatic attraction over the number of reporters that adhere to the solid support beads. This is most clearly demonstrated by the γ-irradiation experiments. Creation of a “hairy” polyelectrolyte coating on reporters by cross-linking the coating in polyelectrolyte solution dramatically increases the number of reporters that adhere to support beads, even though the ξ-potential of the reporters decreases. The polyelectrolyte “tendrils” that extend from the reporter into solution appear to interact with the pendant poly(ethylene glycol) (PEG) chains on the PEGA support bead to form a permanent interlocking molecular web. In contrast, reporters that are coated with a compact cross-linked network adhere in low numbers. Inhibition of the polyelectrolyte chain mobility occurs as the radiation dose is
Encoding Combinatorial Libraries
increased, and the decrease in mobility limits the interaction of the polyelectrolytes with the PEG chains of the support bead. This hinders the formation of a secure attachment to the PEGA bead, and the reporters adhere in numbers that abruptly decrease, as the mesh becomes more compact (i.e. the radiation dose is increased). In summary, a new application of silica colloids that facilitates encoding of large combinatorial libraries has been presented. It is necessary to control the number of reporters that adhere per step to ensure that there are sufficient reporters attached for unambiguous decoding but not too many present such that reporter adhesion in later cycles is inhibited. It is equally important to achieve robust adhesion of the reporters to the bead. This would
Langmuir, Vol. 16, No. 25, 2000 9715
allow fewer reporters to be used in the bar code and reduce the potential for cross-contamination. The process of silica colloid adhesion to solid support beads that has been demonstrated here indicates that the colloidal bar coding strategy is an extremely attractive and economical route to encoding large chemical libraries. Acknowledgment. This work was supported by the Australian Research Council (98/ARCL227). Preliminary experimental work was performed by Daniel Matthews as part of his Honors studies, and we acknowledge the inclusion of his scanning electron microscope image. LA000995Z
