A universal method for the functionalization of dyed magnetic microspheres with peptides

Methods 158 (2019) 12–16

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A universal method for the functionalization of dyed magnetic microspheres with peptides

T

Matthew B. Coppock , Dimitra N. Stratis-Cullum ⁎

Sensors and Electron Devices Directorate, U.S. Army Research Laboratory, 2800 Powder Mill Rd, Adelphi, MD, United States

ARTICLE INFO

ABSTRACT

Keywords: Dyed microspheres Luminex Peptide Multiplex Bead-based assay

The need for the functionalization of magnetic, water-soluble dyed microspheres with peptides is apparent with the ever-growing biointeraction capabilities and the increased use of dyed microspheres in multiplex, microsphere-based detection assays. This method describes the attachment of any peptide to dyed magnetic microspheres regardless of peptide length, size, or sequence. The method exploits ‘click’ chemistry with short reaction times in a mixed organic/water system for simultaneous selective surface functionalization and reduction of microsphere dye leaching. All optimization studies were performed using a Luminex 200 assay platform, but the functionalized microspheres are capable of use in any similar multiplex format.

1. Introduction Monoclonal antibodies (mAbs) are the gold standard reagents for biological detection applications due to their high affinities and high selectivities towards antigens of interest. However, certain properties inherent to mAbs such as low thermal stability, lengthy production times, and batch-to-batch variability, reduces their consistent functionality in devices used outside of a controlled environment and their preparation against emerging threats. As a result, numerous antibody alternative technologies have been developed [1]. One promising alternative is the discovery and maturation of peptide-based reagents. Peptides are advantageous due to their high thermal stabilities which could help eliminate cold chain transport, improve long-term storage, and allow at-point detection in austere environments. Peptides also allow on-demand scalability (grams of material at a time) through robotic methods and are easily modified with non-natural functionalities and tags through synthetic methods for assay integration, sometimes from the onset of discovery. Large biological capture agents, such as mAbs, are typically attached to surfaces through amide chemistry with the N-terminus of the biomolecule or the amino acid lysine presented on its surface. While amide linking is possible with the N-terminus of a peptide, attachment through lysines in the structure could have a profound effect on the activity of the reagent since peptides are such short sequences. Lysine

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side chains could potentially be protected to prevent undesired reactions, but this adds unnecessary complexity to the functionalization procedure. Therefore, specific modifications can be made to either termini of the peptide to provide precise control of peptide presentation on the surface. ‘Click’ chemistry, for example, can be exploited to achieve this precise attachment of biomolecules to various surfaces [2]. Cycloaddition chemistry for the attachment of peptides, as substitutes for mAbs, onto solid supports can potentially be a powerful method for the preparation of thermally stable biological assays such as lateral flow assays and immunoassays like latex agglutination [3]. With multiplex assay capabilities becoming more popular, the functionalization of dyed microspheres is important. For example multiplex platforms produced by companies such as Luminex can test up to 50–100 different analytes simultaneously through sandwich-based assays using modified dyed microspheres [4]. The Luminex instruments are of high interest because they can be found in some mobile lab settings and are more sensitive compared to other similar devices [5]. While antibodies are typically used for capture receptors on the bead surface in such an embodiment, peptides are also capable of performing as capture receptors. The need for the functionalization of dyed magnetic microspheres with peptides is apparent with the ever growing capabilities of peptide reagents and the increased utilization of dyed magnetic microspheres in multiplex detection assays. ‘Click’ chemistry has been previously used

Corresponding author. E-mail address: [email protected] (M.B. Coppock).

https://doi.org/10.1016/j.ymeth.2019.01.014 Received 23 July 2018; Received in revised form 20 December 2018; Accepted 23 January 2019 Available online 30 January 2019 1046-2023/ Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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M.B. Coppock and D.N. Stratis-Cullum

to functionalize particles, including a mixed organic/water solvent system used for organic soluble nanoparticle functionalization [6], but there is a specific need for a method to functionalize water-soluble, dyed microspheres which restricts the leaching of the incorporated dye. Therefore we present a universal method to functionalize dyed microspheres with peptides utilizing single-arm and multi-arm PEG linkers. This method is capable of attaching any peptide sequence or length, which is especially important for short or cyclic peptides that are sometimes difficult to solubilize. Careful optimization was performed to limit dye leaching from the microspheres [7], since an organic solvent is needed to keep the peptide in solution. As a model system, we used a Streptavidin binding peptide (AWRHPQGG) and biotin within the Luminex platform. Successful integration of small hydrophobic peptides with terminal biotins and a 15-mer Abrax (recombinant A-chain of the abrin toxoid) binding peptide [8] provide additional examples of the generality of the method.

setting 5–6, and (5) place tube back on magnetic separator 2.3. Coupling of amine-PEGn-azidez linker to microspheres (Scheme 1A) 2.3.1. Stock solutions

• 1 mg/mL Azido PEG Amine (N3-PEG-NH2), MW 1000 or 4-arm (1•

arm-Amine, 3-arm-Azide), MW 1000 in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) pH = 6.0 0.8 mg/mL 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in 100 mM MES pH = 6.0

2.3.2. Functionalization protocol

• Transfer

2. Materials and methods

• • • • • • •

2.1. Materials MagPlex magnetic COOH microsphere spectrally distinct sets were purchased from Luminex Corp. Azido polyethylglycine (PEG) amine (N3-PEG-NH2), molecular weight (MW) 1000 Da was obtained from Nanocs and 4-arm (1-arm-Amine, 3-arm-Azide), TFA salt, MW 1000 was purchased from Jenkem Technology USA, Inc. Streptavidin conjugated R-Phycoerythrin (SAPE), 1.5 mL Eppendorf Lo-Bind tubes, and phosphate buffered saline (PBS) Protein Free T20 blocker was purchased from ThermoFisher. Recombinant biotinylated Abrax was prepared as previously described [8]. Human serum was obtained from Omega Scientific. Rink amide resin and Fmoc-L-amino acids were acquired from Aapptec. L-Propargylglycine (Pra) functionalized Abrax binding peptide A05 (Pra-PLSFGYWAALEECALC) was purchased from Peptide 2.0. All solvents were purchased from Alfa Aesar, and all other chemicals were purchased from Sigma Aldrich.

80 µL of a MagPlex microsphere bead set (1.25 × 107 beads/mL) to a 1.5 mL Eppendorf Lo-Bind tube, placed on a microcentrifuge magnetic separator for 30 sec, and the liquid was removed Wash microspheres 1× with 250 µL deionized (DI) water followed 1× with 250 µL 100 mM MES pH = 6.0 Resuspend microspheres in 80 µL 0.1 MES pH = 6.0 Dilute 120 µL Amine-PEGn-Azidez stock in 880 µL 100 mM MES pH = 6.0 (1 mL total solution) and add to microspheres Add 200 µL EDC for a total volume of 1280 µL in tube Rotate tube at room temperature in the dark for 2 h Wash microspheres 2× with 250 µL DI water Resuspend microspheres in 80 µL DI water

2.4. Addition of peptides to microspheres via ‘click’ reaction (Scheme 1B) 2.4.1. Stock solutions

• 20 mM Copper(II) Sulfate (CuSO ) in DI water • 50 mM tris-hydroxypropyltriazolylmethylamine water • 200 mM Sodium Ascorbate in DI water • 1 mM Pra-peptide in dimethyl sulfoxide (DMSO) • 50% DMSO in DI water 4

2.1.1. Peptide synthesis and N-terminal functionalization The Streptavidin binding peptide (AWRHPQGG; Fig. S1A) was purchased on resin from Peptide 2.0. The more hydrophobic peptides (Pra-Cy(AFHFY) (Fig. S2A) and Pra-Cy(WNQVW) (Fig. S3A)) were synthesized on Rink Resin in an Aapptec Titan 357 peptide synthesizer and cyclized through established methods [9]. Cy() = triazole cyclization via flanking Pra and L-azidolysine (Az4) residues. The N-terminus of all peptides were functionalized with Pra under typical peptide coupling conditions. After resin cleavage with 95:5 Trifluoroacetic Acid (TFA):Water for 2 h and cold ether precipitation, the peptides were purified on a Shimadzu preparatory scale high performance liquid chromatography (HPLC) instrument with a Phenomenex Jupiter 5u C18 column running a 20%-70% gradient acetonitrile in water over 1 h. The structures were verified by mass spectrometry (Fig. S1B–S3B).

(THPTA) in DI

2.4.2. Functionalization protocol

• Sonicate 80 µL azide functionalized microspheres for 1 min • Add the following solutions in order to 80 µL azide functionalized 1) 2) 3) 4)

• • •

2.2. General considerations

• The protocol was optimized for 1,000,000 Luminex MagPlex COOH magnetic microspheres • Functionalization protocols were modified from existing methods [10,11] • All washing steps follow a general protocol: (1) Place tube with

• • • •

magnetic microspheres on magnetic separator, (2) remove liquid from tube, (3) add wash liquid, (4) vortex tube for ∼ 5–10 sec at

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microspheres 127.5 µL 50% DMSO in DI water 10 µL 1 mM Pra-peptide in DMSO 7.5 µL ‘click’ solution (pre-mix 2.5 µL CuSO4 + 5 µL THPTA) 25 µL Sodium Ascorbate Vortex tube for 5–10 s Rotate tube for 1 h at room temperature in the dark Remove liquid and incubate microspheres 2× with 250 µL 5% Sodium diethyldithiocarbamate trihydrate in DI water rotating at room temperature for 5 min to chelate any free copper Wash microspheres 2× with 250 µL DI water and 2× with 250 µL PBS Protein Free T20 Resuspend microspheres in 500 µL PBS Protein Free T20 Block microspheres for 30 min at room temperature, or store in refrigerator overnight Count number of microspheres in solution using a cell counter (count should be around 2,000,00 in 500 µL of liquid)

Methods 158 (2019) 12–16

M.B. Coppock and D.N. Stratis-Cullum

Scheme 1. Two-step scheme for MagPlex microsphere functionalization with peptides.

2.5. Luminex assay

of the alkynated peptide (1 mM) is utilized to ensure the complete reaction of all free azides available on the surface of 1,000,000 beads. The incorporation of DMSO to the protocol is imperative to guarantee complete solubilization of any peptide regardless of size and hydrophobicity. Unfortunately, organic solvents like DMSO are capable of inducing dye leaching from the microspheres, resulting in reduced assay sensitivity [7]. Again, using alkyne-biotin for an initial investigation, the effect of increasing concentrations of DMSO was tested over 1 h and 24 h. The leaching seems to be fairly DMSO concentration independent, but there is a 20–30% loss in signal over 24 h versus 1 h, as shown in Fig. 1B. A 1 h reaction time in 50% DMSO in water were the optimized conditions to warrant reduced dye leaching and complete solubility of any substrate for functionalization. Once the peptide is attached to the microsphere, everything is completely soluble in buffer. In order to optimize the median fluorescent intensity (MFI) signal of the assay, six different lengths of amine-PEGx-azide (x = 6, 10, 17, 34, 58, and 169) were used to modify the microspheres. The addition of a streptavidin binding peptide (AWRHPQGG) [12] to each functionalized surface, performed in order to show that peptides remain active after attachment, resulted in a fairly Gaussian linker effect. We suspect that the shorter linker lengths reduce the number of analytes capable of bead binding due to steric implications as multiple copies of the analyte compete for surface binding and the longer linker lengths are much more prone to entanglement. It is apparent, as shown in Fig. 1C, that the amine-PEG17-azide (MW 1000) provides the ideal distance for the most binding events to occur resulting in the highest signal. This linker length was used in all subsequent studies and consistently achieves MFI saturation ∼25000–32000 depending on the peptide sequence. An additional PEG linker experiment was performed to attempt to improve the limit of detection (LOD) for streptavidin with AWRHPQGG functionalized microspheres in a Luminex assay. A 4-arm PEG construct (1 amine and 3 azide) was similarly attached to the microsphere surface before addition of alkynated AWRHPQGG. By increasing the number of AWRHPQGG peptides on the microsphere surface by 3-fold, the detection limit and EC50 were improved by 5-fold (mono-arm: EC50 = 493 ± 5 ng/mL; multi-arm: EC50 = 87 ± 1.2 ng/mL), as shown Fig. 2A. The incorporation of various numbers of PEG-azide arms on the microsphere surface is a potential method for tuning binding affinities to fit the needs of an assay. It was also encouraging that even with the increased peptide surface coverage, the microspheres

All Luminex assays were performed and analyzed as described in the Luminex xMAP Cookbook 2nd Edition [10]. Single point experiments involving biotin were performed with 5 µg/mL SAPE in 1:10 PBS Protein Free T20 in PBS, while all full curve experiments with AWRHPQGG functionalized beads were serially diluted for a total of 12 concentrations ranging from 10 µg/mL to 0.0049 µg/mL of SAPE in 1:10 PBS Protein Free T20 in PBS, 1% Human Serum in 1:10 PBS Protein Free T20 in PBS, or 5% Human Serum in 1:10 PBS Protein Free T20 in PBS depending on the experiment. Single point Abrax experiments incubated 12.5 µM biotinylated recombinant Abrax in PBS Protein Free T20 for 2 h at room temperature with the peptide functionalized beads, followed by a 45 min incubation with 5 µg/mL SAPE after washing. Every other assay was allowed to incubate overnight at 4 °C with agitation. All wells were exchanged with 100 µL Luminex Sheath Fluid before injecting into the Luminex 200 analyzer. 3. Results and discussion Various experiments were performed in order to achieve optimal functionalization of the microsphere surface with peptides. During the first step (Scheme 1A), an extreme excess of amine-PEG-azide linker was used to ensure complete coverage of 1,000,000 microspheres with the azide ‘click’ handle. These conditions should actually be capable of total microsphere coverage for more than 1,000,000 microspheres. It is important to note that even though this method was performed with azide functionalized microspheres and alkynated peptide, the same procedure can be followed with alkynated microspheres and azidated peptide, although some studies have suggested that having the surface functionalized with azide is the most efficient [2]. As an initial test to determine the ideal concentration of alkynated substrate for the ‘click’ reaction to occur on microsphere (Scheme 1B), alkyne-biotin was used due to its high affinity for streptavidin. In fact, the bioconjugate SAPE is used during the final incubation for detection in most Luminex assays. Several equivalents, specifically 5×, 10×, 50×, and 100× of alkynebiotin were tested against 100,000 azidated microspheres with an excess of CuSO4, THPTA, and sodium ascorbate. As seen in Fig. 1A, ∼1 µM of the alkynated substrate was sufficient for the functionalized microspheres to achieve maximum signal. As a result, a 1000× excess 14

Methods 158 (2019) 12–16

M.B. Coppock and D.N. Stratis-Cullum

Fig. 1. (A) Concentration optimization for alkynated substrates for microsphere functionalization. (B) DMSO effects on signal output after 1 h and 24 h ‘click’ reaction times. (C) MFI signal produced as a result of PEG azide linker length.

were still highly selective for streptavidin in a matrix of various human serum concentrations (Fig. 2B). Incorporation of the multi-arm constructs requires assay optimization since the increased avidity could actually reduce peptide selectivity in higher serum concentrations which are sometimes present in diagnostic assays. The optimized protocol was then applied to the functionalization of dyed microspheres with biotinylated cyclic peptides (cy(AFHFY) and cy (WNQVW)), as well as a 15-mer Abrax binding peptide [8], shown in

Fig. 3. The cyclic peptides are representative of small, hydrophobic sequences that are difficult to solvate in pure water or buffer. After successful attachment to the microspheres, the signal was comparable to the positive control microspheres functionalized strictly with biotin. A longer, more charged Abrax binding peptide (A05) was also attached to the magnetic microspheres. The 15-mer peptide sequence includes multiple glutamic acid residues, along with two cysteine amino acids. As a capture reagent on the bead surface, A05 successfully detected

Fig. 2. Streptavidin detection with AWRHPQGG using the (A) single azide linker (red) versus the multi-azide linker (blue). (B) Detection of streptavidin out of buffer (black), 1% human serum (green), and 5% human serum (red) with multi-arm AWRHPQGG microspheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 15

Methods 158 (2019) 12–16

M.B. Coppock and D.N. Stratis-Cullum

Fig. 3. Signal detected after successful integration of small, hydrophobic, biotinylated cyclic peptides (AFHFY and WNQVW) and an Abrax binding peptide (A05) onto the microsphere surface compared to biotinylated microspheres (Pos), bare microspheres (Neg 1), azide functionalized microspheres (Neg 2), “reacted” microspheres without the copper catalyst (Neg 3), and SAPE only with A05 (A05(neg)).

doi.org/10.1016/j.ymeth.2019.01.014.

Abrax in the sandwich-based assay and exhibited high selectivity for Abrax over streptavidin (Fig. 3) similar to previously published result on cell [8]. In these cases, we are simply showing that peptides are capable of replacing antibodies as receptors. Since the binding performance of peptide reagents varies from peptide to peptide, it is necessary to perform similar binding experiments when developing new multiplex assays.

References [1] Vincent J.B. Ruigrok, M. Levisson, Michel H.M. Eppink, H. Smidt, J. van der Oost, Alternative affinity tools: more attractive than antibodies? Biochem. J 436 (2011) 1–13. [2] X.-L. Sun, C.L. Stabler, C.S. Cazalis, E.L. Chaikof, Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions, Bioconjug. Chem. 17 (2006) 52–57. [3] L.B. Bangs, Recent uses of microspheres in diagnostic tests and assays, in: Z. Liron, A. Bromberg, M. Fisher (Eds.), Novel Approaches in Biosensors and Rapid Diagnostic Assays: 43rd OHOLO Conference Eilat, Israel, October 10–14, 1999, Springer, US, Boston, MA, 2001, pp. 245–263. [4] S.A. Dunbar, M.R. Hoffmeyer, Microsphere-Based Multiplex 2.9 Immunoassays: Development and Applications Using Luminex® xMAP® Technology, The Immunoassay Handbook: Theory and applications of ligand binding, ELISA and related techniques, (2013) 157. [5] J. Betters, R. Dorsey, P. Emanuel, B. Rivers, E. Schaffer, E. Skowronski, Edgewood Biosensors Test Bed Hand-held and Man-Portable Edition, DTIC Document, 2013. [6] M.A. White, J.A. Johnson, J.T. Koberstein, N.J. Turro, Toward the syntheses of universal ligands for metal oxide surfaces: controlling surface functionality through click chemistry, J. Am. Chem. Soc. 128 (2006) 11356–11357. [7] D.J. Chandler, Encapsulation of discrete quanta of fluorescent particles, Google Patents, 2003. [8] D.A. Sarkes, M.M. Hurley, M.B. Coppock, M.E. Farrell, P.M. Pellegrino, D.N. StratisCullum, Rapid discovery of peptide capture candidates with demonstrated specificity for structurally similar toxins, 2016, pp. 986305-986305-986310. [9] S. Das, A. Nag, J. Liang, D.N. Bunck, A. Umeda, B. Farrow, M.B. Coppock, D.A. Sarkes, A.S. Finch, H.D. Agnew, S. Pitram, B. Lai, M.B. Yu, A.K. Museth, K.M. Deyle, B. Lepe, F.P. Rodriguez-Rivera, A. McCarthy, B. Alvarez-Villalonga, A. Chen, J. Heath, D.N. Stratis-Cullum, J.R. Heath, A general synthetic approach for designing epitope targeted macrocyclic peptide ligands, Angew. Chem. Int. Ed. 54 (2015) 13219–13224. [10] S. Angeloni, R. Cordes, S.A. Dunbar, C. Garcia, G. Gibson, C. Martin, V. Stone, xMAP Cookbook, 2nd, Edition, 2014. [11] G. Research Product – THPTA – A Water Soluble Click Ligand 15 (May New 2017). [12] M.H. Caparon, P.A. De Ciechi, C.S. Devine, P.O. Olins, S.C. Lee, Analysis of novel streptavidin-binding peptides, identified using a phage display library, shows that amino acids external to a perfectly conserved consensus sequence and to the presented peptides contribute to binding, Mol. Divers. 1 (1996) 241–246. [13] A.E.M. Wammes, M.J.E. Fischer, N.J. de Mol, M.B. van Eldijk, F.P.J.T. Rutjes, J.C.M. van Hest, F.L. van Delft, Site-specific peptide and protein immobilization on surface plasmon resonance chips via strain-promoted cycloaddition, Lab Chip 13 (2013) 1863–1867.

4. Conclusions The described method allows functionalization of dyed magnetic microspheres under mild reaction conditions with peptides regardless of sequence or length. Unless the microspheres are specifically encapsulated to be compatible with organic solvents, dye leaching from the microspheres is known to occur in these solvents over a prolonged period of time [7]. Peptides, on the other hand, exhibit varying degrees of solubility in aqueous conditions depending on the length and sequence, and often require organic solvents to dissolve. A multi-arm linker attached to the microsphere surface has the ability to improve a modest binder into a much more useable detection reagent. While the method described requires a Cu(I) catalyst, the same result could be achieved catalyst-free using strain-promoted cycloaddition reagents like cyclooctynes [13]. Additionally, the protocol could be performed on non-magnetic dyed microspheres, but with all washing steps needing to be performed by centrifugation or within spin filter tubes. Acknowledgements Funding: This work was partially supported by the Institute for Collaborative Biotechnologies from the U.S. Army Research Office [grant number W911NF-09-D-0001]. The content of the information does not necessarily reflect the position of the policy of the United States Government, and no official endorsement should be inferred. Appendix A. Supplementary data Supplementary data to this article can be found online at https://

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