IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 61, NO. 3, MARCH 2014
705
Peptide Synthesis on Glass Substrate Using Acoustic Droplet Ejector Youngki Choe∗ , Student Member, IEEE, Shih-Jui Chen, Member, IEEE, and Eun Sok Kim, Fellow, IEEE
Abstract—This paper describes the synthesis of a 9-mers-long peptide ladder structure of glycine on a modified glass surface using a nanoliter droplet ejector. To synthesize peptide on a glass substrate, SPOT peptide synthesis protocol was followed with a nozzleless acoustic droplet ejector being used to eject about 300 droplets of preactivated amino acid solution to dispense 60 nL of the solution per mer. The coupling efficiency of each mer was measured with FITC fluorescent tag to be 96%, resulting in net 70% efficiency for the whole 9-mer-long peptide of glycine. Usage of a nanoliter droplet ejector for SPOT peptide synthesis increases the density of protein array on a chip. Index Terms—Droplet ejector, nozzleless droplet ejector, peptide synthesis, protein synthesis, SPOT synthesis.
I. INTRODUCTION EPTIDE synthesis is traditionally conducted in liquid phase. However, in applications that require peptide array on a solid substrate, liquid-phase synthesis is not sufficient, since peptide has to interact with solid surface. In solid phase peptide synthesis (SPPS) pioneered by Merrifield [1], small solid beads (or resins) usually made of porous glass are treated with linkers on which peptide chain is built. The synthesized peptide stays on the beads until it is chemically cleaved by a reagent. Since the overall yield of peptide synthesis is directly dependent on the stepwise yield of adding single mer of amino acid, it is very important to maximize the stepwise yield. For example, though the final yield for 26-mer-long peptide synthesis would be 77% for a 99% stepwise yield, it would drop to 25% when the stepwise yield decreased to 95%. For this reason, SPPS chemistry has been improved for a higher stepwise yield in resins [2] and linkers [3]–[5]. As the original SPPS protocol synthesizes one kind of peptide at a time, a new technique called SPOT synthesis technique was introduced by Frank [6] for in situ parallel peptide synthesis on a membrane. SPOT technique uses membrane, rather than beads, on which peptide is synthesized, but follows basically the
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Manuscript received January 13, 2013; revised May 7, 2013; accepted May 25, 2013. Date of publication October 24, 2013; date of current version February 14, 2014. This work was supported by the National Institutes of Health under Grant R21HG005118. Asterisk indicates corresponding author. ∗ Y. Choe is with the Department of Electrical Engineering—Electrophysics, University of Southern California, Los Angeles, CA 90089 USA (e-mail: [email protected]). S.-J. Chen and E. S. Kim are with the Department of Electrical Engineering— Electrophysics, University of Southern California, Los Angeles, CA 90089 USA (e-mail: [email protected]; [email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TBME.2013.2287218
same principle and chemistry of SPPS. With SPOT technique, different sequences of peptides can easily be synthesized on multiple spots in parallel on a solid surface [7]–[11]. In conventional SPOT peptide synthesis on cellulose membranes performed with array of micropipette, the spot size varies from several millimeters to few centimeters in diameter because of the large amount of the dispensed chemicals and also the chemicals’ spreading. A SPOT synthesis was demonstrated on a modified glass surface that was free from the liquid spreading issue [12]. High-density peptide array can be formed on a glass slide, as the dispensed liquid keeps its boundary without spreading, with a nanoliter liquid dispenser capable of precise volume control. This paper describes our recent synthesis of glycine peptides with various molecular lengths (1–9 mers) on a modified glass surface using SPOT peptide synthesis protocol. Using an acoustic ejector, we have reduced the spot size for peptide synthesis by dispensing droplets on photolithographically defined active spots on a glass substrate where the dispensed liquid does not spread out much. With the reduced spot size on a solid substrate, the key technical barrier to obtain a highly packed protein array is in how small the dispensing amount can be reduced. The acoustic ejector offers a very good means to dispense small amount of liquid with precise volume control, since it does not require a nozzle. An acoustic ejector employing silicon Fresnel lens dispensed nanoliter droplets of preactivated amino acid solution onto active spots with 1 mm diameter on a glass substrate. The stepwise coupling yield was measured to be 96%, and the final yield of a 9-mers-long peptide synthesis was measured to be 70% when estimated from the measured light intensity coming from FITC fluorescent tag. II. PROCEDURE FOR PEPTIDE SYNTHESIS WITH DROPLET EJECTOR A. Surface Modified Glass Preparation To prepare a surface modified glass surface, a glass slide was first cleaned with piranha solution (four parts of H2 O2 and 1 part of H2 SO4 ) for 30 min. Then, the glass slide was cleaned with acetone (three times) and chloroform (three times), consecutively, by submerging the slide in a beaker which contains about 20 mL of either acetone or chloroform and shaking the beaker for 60 s. To define the active spots (see Fig. 1) on which amino acids will be attached later, AZ 5214 photoresist was spin-coated onto a glass slide at 1500 rotations per minute (r/min), and exposed with a photomask on a mask aligner. After developing the photoresist, the glass slide was submerged in 1% (v/v) 1H,1H, 2H, 2H–perfluoredecylmethyldichlorosilane in
0018-9294 © 2013 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications standards/publications/rights/index.html for more information.
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Fig. 1.
IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 61, NO. 3, MARCH 2014
Preparation steps for the amine-terminated glass surface.
Fig. 2. Photos of (a) top view of the packaged device and (b) the ejector before the addition of the top cover.
2,2,4-trimethyl-pentane solution for 15 min to form a hydrophobic perfluorinated layer. After washing the glass slide five times with n-hexane and three times with chloroform, the photoresist was completely removed with acetone. The spot-arrayed glass was submerged in 5% (v/v) γ-APTS in chloroform solution at 50 ◦ C for 1 h to silanize the spots and to form direct amine. After washing the glass slide five times with chloroform, we dried the glass slide in vacuum for 1 h before dispensing the first amino acid solution onto the glass surface with an acoustic droplet ejector. B. Ejector Design and Fabrication The acoustic ejector mainly consists of an acoustic transducer and a lens with air-reflectors (see Fig. 3) was designed. The 127 μm thick PZT sheet (with the fundamental thicknessmode resonant frequency of 18 MHz) sandwiched between two nickel electrodes serves as the acoustic transducer. A micromachined silicon is used as the air-cavity-lens structure. The acoustic waves produced by the PZT propagate into water through the silicon, but are mostly reflected by the air pockets (since the acoustic impedance of air is comparatively infinitesimal). The air-cavity lens is patterned into annular Fresnel half-wave bands with a focal length F of 800 μm with the radius of the kth
Fig. 3. Brief fabrication steps of the acoustic droplet ejector with silicon lens. (a) bulk-micromachining to make front-backside alignment mark. (b) dry etching to form trenches with Fresnel lens pattern after removing SiNx . (c) dry etching forming ejecting chamber, liquid transfer channel, and reservoir. (d) PZT transducer with 0.4 μm thick top and bottom nickel electrodes. (e) PZT transducer and silicon lens combined with low viscosity glue.
Fresnel band being designed according to the equation shown in [13]. The fabrication steps are described below. First, a 3 silicon wafer (deposited with LPCVD SiNx ) was bulk-micromachined (with KOH) to make front-backside alignment marks using the patterned SiNx as an etch mask during KOH etching of silicon [see Fig. 3(a)]. After removing the SiNx with hot phosphoric acid, the front side of the silicon wafer was dry-etched in deep reactive ion etcher (DRIE) to form 20 μm deep trenches with Fresnel lens pattern [see Fig. 3(b)]. The backside also was dry-etched to form ejection chambers, liquid transfer channels, and liquid reservoirs [see Fig. 3(c)]. A PZT substrate was processed separately by depositing and patterning 0.4 μm thick top and bottom nickel electrodes over a 127 μm thick PZT sheet [see Fig. 3(d)]. Low viscosity glue with viscosity of 150 Hz was spin-coated onto the PZT transducer at 6000 r/min, and the PZT transducer was glued to the silicon wafer [see Fig. 3(e)]. The glue was cured in elevated temperature of 80 ◦ C for 24 h. After the integration of the silicon lens and the PZT transducer, another bulk-micromachined silicon wafer
CHOE et al.: PEPTIDE SYNTHESIS ON GLASS SUBSTRATE USING ACOUSTIC DROPLET EJECTOR
Fig. 4. Conceptual steps on how droplets of amino-acid are coupled onto the glass substrate: (a) free amine on the glass surface, (b) coupling of preactivated amino-acid to free amine on the glass, (c) capping of uncoupled free amines, (d) fmoc-deprotection for next mer coupling. (e1) coupling next amino acid. (e2) coupling FITC for optical measurement.
Fig. 5. spots.
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Synthesis of peptide ladders of nine different lengths over nine different
D. Synthesis of Peptide Ladder was added to act as a top cover providing a mechanism to maintain the liquid level constant. Then, the completed ejector was mounted on a dual-inline-package (DIP) socket [see Fig. 2(a)]. The dimension of the fabricated acoustic ejector is measured to be 6 mm (width) × 12 mm (length) × 1 mm (thickness). The ejector consists of ejecting chamber, liquid transfer channel, and liquid reservoir. The ejecting chamber, in which acoustic lens-with-air-reflector is sitting, occupies 3 mm (width) × 3 mm (length) × 1 mm (thickness). The dimensions of the liquid transfer channel and reservoir can easily be reduced or avoided by using other liquid supply techniques such as a tube and/or liquid cartridge. C. Coupling Amino-Acid Onto the Glass Surface To couple amino acids, 60 nL (corresponding to 300 droplets of 0.2 nL) of the preactivated solution (6.7 mM) of fmoc-amino acid with 6.7 mM of BOP, HOBt, and DIPEA in N-methyl2-pyrrolidone (NMP) was dispensed using an acoustic droplet ejector onto the amine-terminated spots on the surface modified glass slide, and let to react for 60 min at room temperature [see Fig. 4(b)]. The uncoupled free amine groups in the active spots were capped with 10% (v/v) solution of acetic anhydride and pyridine (1:1) in NMP for 30 min at room temperature [see Fig. 4(c)]. The fmoc-protection group was then removed with 20% (v/v) solution of diethylamine in NMP for 30 min at room temperature to liberate free-amine group [see Fig. 4(d)]. The liberated free-amine group was coupled with next mer aminoacid [see Fig. 4(e1)] or reacted with a 5.0 mM solution of FITC in NMP for 2 h at room temperature to attach FITC fluorescent tag at the end of the peptide for an optical measurement that quantifies the number of the coupled amino acid [see Fig. 4(e2)].
A peptide ladder consisting of glycine from 1-mer to 9-mers was synthesized on nine different spots on a surface modified glass slide (see Fig. 5). At the start of the synthesis, about 60 nL of preactivated amino-acid solution was dispensed onto all the nine active spots using the fabricated acoustic droplet ejector. The glass slide was placed about 2 mm right above the opening of the ejecting chamber (through which droplets are vertically ejected). After dispensing 60 nL of the solution on the desired spot, the glass slide was moved so that the ejector can dispense the solution onto the next active spot. Once the ejector dispenses the preactivated amino acid solution onto all of the nine active spots, the active spots were left for 60 min at room temperature for reaction with the dispensed amino acid to occur. After washing the whole glass slide two times with NMP and one time with methyl alcohol, we blow-dried the glass slide with dry nitrogen. The uncoupled free-amine groups in the active spots were capped for 30 min by dispensing 200 nL of capping solution with a pipette. Then, after washing out the capping solution, we removed fmoc-protection group from eight active spots (out of the nine total spots) by dispensing 200 nL fmocdeprotection solution, so that the eight spots would be ready to be coupled with next-mer amino acid. At this moment, the ninth spot was not active anymore, and it had only 1-mer-long glycine peptide to the end of the experiment. After washing the fmoc-deprotection solution, we moved the glass slide back onto the ejector for dispensing next mer amino acid. By repeating the steps above, all the nine spots had different lengths of peptide from 1 to 9 mers (see Fig. 5). When all the nine spots had desired number of peptides in the ladders, fmoc-protection groups of all the nine spots were removed so that FITC might be attached to the synthesized peptides (see Fig. 6). After FITC attachment, the light intensities
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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 61, NO. 3, MARCH 2014
Fig. 7. Experimental setup for ejecting droplets of preactivated amino-acid solution onto a surface-modified glass slide. Fig. 6. FITC attachment at the end of the peptide chains after the synthesis of the peptide ladders.
coming from the nine spots were measured with a fluorescent microscope at 532 nm. It took about 3 h to add 1-mer to a peptide chain with the technique used in the 9-mer peptide synthesis, mainly because the time demanding steps of washing, drying, reacting between chemicals. Also, preparing a bare glass slide to be ready for coupling with the first peptide mer took us about 2 days, because of cleaning any possible organic contamination, surface modification, washing in between surface modification steps, and drying in vacuum. However, with automation and optimization, the time can be reduced substantially. III. MEASUREMENT SETUP A. Droplet Ejection Setup The fabricated ejector was actuated with pulsed 17.3 MHz sinusoidal signals at 60 Hz pulse repetition frequency. The pulsed signal was amplified with an RF power amplifier to drive the ejector with a voltage amplitude of 160 Vp eak -to -p eak . A red LED was used as a light source to stroboscopically observe the ejection process through adjusting the delay between the device actuation and LED illumination (see Fig. 7). A chargecoupled device (CCD) camera being attached at the end of a long-working-range microscope was placed horizontally to record the ejection process to a computer. The glass slide with free-amine groups on its active spots was placed about 2 mm away from the ejector and aligned, so that the ejected droplets of preactivated amino-acid solution can ink the active spots of the glass slide. B. Light Intensity Measurement Setup A fluorescent microscope equipped with cooled CCD camera was used to detect the optical signals coming from FITC at 532 nm to confirm the synthesis. The cooled CCD camera captured images with exposure time of 30 s, and the captured images were transferred to a computer, and stored in lossless
image format (i.e., TIFF) to avoid data distortion. The light intensity was measured and quantified from the stored image using ImageJ software. IV. MEASUREMENT RESULTS A. Optimum Coupling Time Measurement The longer the coupling time, the better is the coupling efficiency (up to a certain time) in general. But the shorter the coupling time, the better is the throughput of the synthesis. To find out the optimal (or minimum) amino-acid coupling time in our experimental setup, 1-mer glycine was synthesized on a slide glass using a pipette with various reaction times (5, 10, 20, 30, 40, 60, and 90 min). All spots were reacted with FITC solution when the amino-acid coupling is finished. Since the light intensity coming from FITC labeled aminoacid represents the amount of the coupled amino-acid onto glass surface, the light intensity was measured as a function of the coupling time. The measured light intensities were normalized with the following equation, and plotted in Fig. 8 Inorm alized =
In I90 m in − Icapping
(1) only
where In , I90 m in , and Icapping only are the light intensities of the spots treated with n min coupling time, 90 min coupling time, and only with capping solution, respectively. As we can see in Fig. 8, the average light intensity of the spot with 60 min coupling time showed negligible difference from that of the spot with 90 min coupling time. Since the coupling did not improve when the reaction time was longer than 60 min, we fixed the amino-acid coupling time to 60 min in all the following experiments. B. Ejecting Condition Calibration The optimal driving condition for the droplet ejector such as the pulse width and actuation voltage depends on the liquid type. A 17.351 MHz pulsed sinusoidal signal with 2 μs pulse
CHOE et al.: PEPTIDE SYNTHESIS ON GLASS SUBSTRATE USING ACOUSTIC DROPLET EJECTOR
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Fig. 10. Photos showing the collected droplets of preactivated amino acid solution ejected by the acoustic ejector with silicon lens. Each liquid bump has 60 nL of the solution.
Fig. 8. Normalized light intensities measured from the amino-acid coupled spots as a function of coupling time.
Fig. 11. Photos of ejected droplets of the preactivated amino-acid solution that are on their way to a glass slide having free amines on its surface. Each photo shows droplet’s position at (a) 150 μs, (b) 450 μs, (c) 700 μs, and (d) 1000 μs after the droplet is ejected from the ejection chamber. Fig. 9. Photo of the droplet of preactivated amino acid solution just before being ejected out of the ejection chamber.
C. Light Intensity Measurement on Peptide Ladder
width was good to eject droplets of DI water. However, the pulse width had to be increased to 6 μs to eject droplets of preactivated amino acid in N-Methyl-2-Pyrrolidone (NMP) solution, since NMP has 1.7 times higher viscosity (1.7 × 10−3 Pa·s) than DI water (1.0 × 10−3 Pa·s). The droplet size of the acoustic ejector is primarily determined by the focal size which in turn depends on the wavelength of the acoustic wave produced from the transducer, and is very consistent for a given viscosity. In a low viscosity liquid such DI water and alcohols, the diameter of the ejected droplet is about the wavelength of the acoustic wave. With high viscosity liquid such as oil and NMP, the diameter of the ejected droplet is usually larger than the wavelength. However, the acoustic intensity level needed to eject droplets also is higher in case of liquid with higher viscosity, and is likely the reason for a larger droplet size. Even when the viscosity changes over a wide range (up to 55 cSt), the size of the ejected droplet varies less than 10%. The ejected droplets of NMP solution had 80 μm diameters as shown in Fig. 9, and we ejected 300 droplets to dispense 60 nL onto each spot on the glass slide (see Fig. 10)
Fluorescent light intensities of the nine spots of the peptide ladder were measured with a fluorescent microscope. Since the coupling efficiency is not 100%, the light intensity coming from the spot having a longer peptide ladder is lower. Comparing the measured light intensities on different spots, we can obtain the stepwise coupling efficiency or overall coupling efficiency. The preactivated amino-acid solution was dispensed onto the nine spots using acoustic ejector (see Fig. 11), and the fluorescent images taken from each spot of the ladder structure are shown in Fig. 12. The synthesis spot, where peptide is to be synthesized, is defined photolithographically, and the outside of the synthesis spot is perfluorinated to prevent the area from being coupled with peptide’s free-amine group. The perfluorinated surface is rough, and the rough surface makes it easy for fluorescent FITC to physically attach (nonspecifically) onto the area (i.e., the outside of the synthesis spot). Since the synthesis spot is well defined by precise photolithography technique, we can easily find where we should look at to measure the fluorescent light intensity solely due to selective binding between FITC and synthesized peptide. Fig. 13 shows monotonous decrease of the light intensity as the length of the synthesized peptide increased from 1-mer to
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IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 61, NO. 3, MARCH 2014
Fig. 12. Fluorescent images of FITC-attached amino-acids that have been synthesized with the amino-acid droplets ejected by the acoustic ejector. Since the coupling efficiency is not 100%, the light intensity decreases as the length of the synthesized protein increases. Higher light intensity represents more population of the protein on the glass surface.
Fig. 13. Measured fluorescence light intensity versus the length of the synthesized protein. The intensity of the 8-mer protein is about 70% (=0.968 ) of that of the 1-mer protein, indicating a stepwise coupling efficiency of 96%.
9-mers if we exclude the light intensities measured from the spots having 2-mer and 3-mer long peptides. The light intensities coming from those spots were lower than the expected possibly because the quality of the free amine groups on those spots was not as good as the other locations. Since most of the spots showed a reasonable light intensity, the average stepwise coupling efficiency and overall coupling efficiency in synthesizing the 9-mer-long glycine peptide were calculated to be 96% and 70%, respectively. The synthesis results shown in Fig. 13 were from one experiment, though we carried out many repeated experiments to master and confirm the peptide synthesis protocol described in [12] both with the acoustic ejectors and conventional pipettes, as we focused on optimizing the acoustic ejector for synthesis of high-density peptide array. V. CONCLUSION This paper describes protein synthesis with various 200 pL peptides that are ejected onto a solid surface by MEMS droplet
ejectors. We have successfully synthesized 9-mers of glycine on modified glass surface using SPOT technique. Protein microarray can be fabricated through either immobilization of presynthesized protein chain or in situ solid-phase synthesis from peptides. SPOT technique is one of the in situ peptide synthesis methods that is cost effective, easy to automate, and has simple experimental procedure. But SPOT has traditionally relied on micropipettes for spotting active areas with peptides, and is limited in its scalability. The acoustic droplet ejector described in this paper, on the other, is scalable, and will allow massive parallel protein synthesis on a solid substrate from peptides. The acoustic microdroplet ejector with air cavities formed by micromachined silicon was fabricated to dispense droplets of 200 pL on demand, and was used to synthesize a 9-mer-long ladder of glycine on a modified glass surface with 96% stepwise coupling efficiency. Though the active spot size was 1 mm in diameter in this preliminary study, the spot size can easily be reduced to a much smaller diameter with a relatively minor optimization effort. REFERENCES [1] R. B. Merrifield, “Solid phase peptide synthesis—Part I. The synthesis of a tetrapeptide,” J. Amer. Chemical Soc., vol. 85, no. 14, pp. 2149–2154, Jul. 1963. [2] A. R. Mitchell, S. B. H. Kent, M. Engelhard, and R. B. Merrifield, “A new synthetic route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenyl acetamidomethyl-resin, an improved support for solid-phase peptide synthesis,” J. Organic Chem., vol. 43, no. 14, pp. 2845–2852, Jul. 1978. [3] S. S. Wang, “p-Alkoxybenzyl alcohol resin and p-Alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments,” J. Amer. Chem. Soc., vol. 95, no. 4, pp. 1328–1333, Feb. 1973. [4] G. R. Matsueda and J. M. Stewart, “A p-Methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides,” Peptides, vol. 2, no. 1, pp. 45–50, Jan. 1981. [5] P. Sieber, “A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method,” Tetrahedron Lett., vol. 28, no. 19, pp. 2107–2111, Jan. 1987. [6] R. Frank, “Spot-synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support,” Tetrahedron, vol. 48, no. 42, pp. 9217–9232, 1992. [7] R. Frank, “The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—Principles and applications,” J. Immunological Methods, vol. 267, no. 1, pp. 13–26, Sep. 2002. [8] N. Heine, T. Ast, J. Schneider-Mergener, U. Reineke, L. Germeroth, and H. Wenschuh, “Synthesis and screening of peptoid arrays on cellulose membranes,” Tetrahedron, vol. 59, no. 50, pp. 9919–9930, Dec. 2003. [9] X. Espanel and R. H. Huijsduijnen, “Applying the SPOT peptide synthesis procedure to the study of protein tyrosine phosphatase substrate specificity: Probing for the heavenly match in vitro,” Methods, vol. 35, no. 1, pp. 64–72, Jan. 2005. [10] H. E. Blackwell, “Hitting the SPOT: Small-molecule macroarrays advance combinatorial synthesis,” Current Opinion Chem. Biol., vol. 10, no. 3, pp. 203–212, Jun. 2006. [11] K. Hilpert, D. F. H. Winkler, and R. E. W. Hancock, “Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion,” Nature Protocols, vol. 2, pp. 1333–1349, May 2007. [12] D. H. Kim, D. S. Shin, and Y. S. Lee, “Spot arrays on modified glass surface for efficient SPOT synthesis and on-chip bioassay of peptides,” J. Peptide Sci., vol. 13, no. 10, pp. 625–633, Oct. 2007. [13] C. Y. Lee, H. Yu, and E. S. Kim, “Acoustic ejector with novel lens employing air-reflectors,” in Proc. 19th IEEE Int. Conf. Micro Electro Mech. Syst., pp. 170–173, 2006.
Authors’ photographs and biographies not available at the time of publication.
705
Peptide Synthesis on Glass Substrate Using Acoustic Droplet Ejector Youngki Choe∗ , Student Member, IEEE, Shih-Jui Chen, Member, IEEE, and Eun Sok Kim, Fellow, IEEE
Abstract—This paper describes the synthesis of a 9-mers-long peptide ladder structure of glycine on a modified glass surface using a nanoliter droplet ejector. To synthesize peptide on a glass substrate, SPOT peptide synthesis protocol was followed with a nozzleless acoustic droplet ejector being used to eject about 300 droplets of preactivated amino acid solution to dispense 60 nL of the solution per mer. The coupling efficiency of each mer was measured with FITC fluorescent tag to be 96%, resulting in net 70% efficiency for the whole 9-mer-long peptide of glycine. Usage of a nanoliter droplet ejector for SPOT peptide synthesis increases the density of protein array on a chip. Index Terms—Droplet ejector, nozzleless droplet ejector, peptide synthesis, protein synthesis, SPOT synthesis.
I. INTRODUCTION EPTIDE synthesis is traditionally conducted in liquid phase. However, in applications that require peptide array on a solid substrate, liquid-phase synthesis is not sufficient, since peptide has to interact with solid surface. In solid phase peptide synthesis (SPPS) pioneered by Merrifield [1], small solid beads (or resins) usually made of porous glass are treated with linkers on which peptide chain is built. The synthesized peptide stays on the beads until it is chemically cleaved by a reagent. Since the overall yield of peptide synthesis is directly dependent on the stepwise yield of adding single mer of amino acid, it is very important to maximize the stepwise yield. For example, though the final yield for 26-mer-long peptide synthesis would be 77% for a 99% stepwise yield, it would drop to 25% when the stepwise yield decreased to 95%. For this reason, SPPS chemistry has been improved for a higher stepwise yield in resins [2] and linkers [3]–[5]. As the original SPPS protocol synthesizes one kind of peptide at a time, a new technique called SPOT synthesis technique was introduced by Frank [6] for in situ parallel peptide synthesis on a membrane. SPOT technique uses membrane, rather than beads, on which peptide is synthesized, but follows basically the
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Manuscript received January 13, 2013; revised May 7, 2013; accepted May 25, 2013. Date of publication October 24, 2013; date of current version February 14, 2014. This work was supported by the National Institutes of Health under Grant R21HG005118. Asterisk indicates corresponding author. ∗ Y. Choe is with the Department of Electrical Engineering—Electrophysics, University of Southern California, Los Angeles, CA 90089 USA (e-mail: [email protected]). S.-J. Chen and E. S. Kim are with the Department of Electrical Engineering— Electrophysics, University of Southern California, Los Angeles, CA 90089 USA (e-mail: [email protected]; [email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TBME.2013.2287218
same principle and chemistry of SPPS. With SPOT technique, different sequences of peptides can easily be synthesized on multiple spots in parallel on a solid surface [7]–[11]. In conventional SPOT peptide synthesis on cellulose membranes performed with array of micropipette, the spot size varies from several millimeters to few centimeters in diameter because of the large amount of the dispensed chemicals and also the chemicals’ spreading. A SPOT synthesis was demonstrated on a modified glass surface that was free from the liquid spreading issue [12]. High-density peptide array can be formed on a glass slide, as the dispensed liquid keeps its boundary without spreading, with a nanoliter liquid dispenser capable of precise volume control. This paper describes our recent synthesis of glycine peptides with various molecular lengths (1–9 mers) on a modified glass surface using SPOT peptide synthesis protocol. Using an acoustic ejector, we have reduced the spot size for peptide synthesis by dispensing droplets on photolithographically defined active spots on a glass substrate where the dispensed liquid does not spread out much. With the reduced spot size on a solid substrate, the key technical barrier to obtain a highly packed protein array is in how small the dispensing amount can be reduced. The acoustic ejector offers a very good means to dispense small amount of liquid with precise volume control, since it does not require a nozzle. An acoustic ejector employing silicon Fresnel lens dispensed nanoliter droplets of preactivated amino acid solution onto active spots with 1 mm diameter on a glass substrate. The stepwise coupling yield was measured to be 96%, and the final yield of a 9-mers-long peptide synthesis was measured to be 70% when estimated from the measured light intensity coming from FITC fluorescent tag. II. PROCEDURE FOR PEPTIDE SYNTHESIS WITH DROPLET EJECTOR A. Surface Modified Glass Preparation To prepare a surface modified glass surface, a glass slide was first cleaned with piranha solution (four parts of H2 O2 and 1 part of H2 SO4 ) for 30 min. Then, the glass slide was cleaned with acetone (three times) and chloroform (three times), consecutively, by submerging the slide in a beaker which contains about 20 mL of either acetone or chloroform and shaking the beaker for 60 s. To define the active spots (see Fig. 1) on which amino acids will be attached later, AZ 5214 photoresist was spin-coated onto a glass slide at 1500 rotations per minute (r/min), and exposed with a photomask on a mask aligner. After developing the photoresist, the glass slide was submerged in 1% (v/v) 1H,1H, 2H, 2H–perfluoredecylmethyldichlorosilane in
0018-9294 © 2013 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications standards/publications/rights/index.html for more information.
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Fig. 1.
IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 61, NO. 3, MARCH 2014
Preparation steps for the amine-terminated glass surface.
Fig. 2. Photos of (a) top view of the packaged device and (b) the ejector before the addition of the top cover.
2,2,4-trimethyl-pentane solution for 15 min to form a hydrophobic perfluorinated layer. After washing the glass slide five times with n-hexane and three times with chloroform, the photoresist was completely removed with acetone. The spot-arrayed glass was submerged in 5% (v/v) γ-APTS in chloroform solution at 50 ◦ C for 1 h to silanize the spots and to form direct amine. After washing the glass slide five times with chloroform, we dried the glass slide in vacuum for 1 h before dispensing the first amino acid solution onto the glass surface with an acoustic droplet ejector. B. Ejector Design and Fabrication The acoustic ejector mainly consists of an acoustic transducer and a lens with air-reflectors (see Fig. 3) was designed. The 127 μm thick PZT sheet (with the fundamental thicknessmode resonant frequency of 18 MHz) sandwiched between two nickel electrodes serves as the acoustic transducer. A micromachined silicon is used as the air-cavity-lens structure. The acoustic waves produced by the PZT propagate into water through the silicon, but are mostly reflected by the air pockets (since the acoustic impedance of air is comparatively infinitesimal). The air-cavity lens is patterned into annular Fresnel half-wave bands with a focal length F of 800 μm with the radius of the kth
Fig. 3. Brief fabrication steps of the acoustic droplet ejector with silicon lens. (a) bulk-micromachining to make front-backside alignment mark. (b) dry etching to form trenches with Fresnel lens pattern after removing SiNx . (c) dry etching forming ejecting chamber, liquid transfer channel, and reservoir. (d) PZT transducer with 0.4 μm thick top and bottom nickel electrodes. (e) PZT transducer and silicon lens combined with low viscosity glue.
Fresnel band being designed according to the equation shown in [13]. The fabrication steps are described below. First, a 3 silicon wafer (deposited with LPCVD SiNx ) was bulk-micromachined (with KOH) to make front-backside alignment marks using the patterned SiNx as an etch mask during KOH etching of silicon [see Fig. 3(a)]. After removing the SiNx with hot phosphoric acid, the front side of the silicon wafer was dry-etched in deep reactive ion etcher (DRIE) to form 20 μm deep trenches with Fresnel lens pattern [see Fig. 3(b)]. The backside also was dry-etched to form ejection chambers, liquid transfer channels, and liquid reservoirs [see Fig. 3(c)]. A PZT substrate was processed separately by depositing and patterning 0.4 μm thick top and bottom nickel electrodes over a 127 μm thick PZT sheet [see Fig. 3(d)]. Low viscosity glue with viscosity of 150 Hz was spin-coated onto the PZT transducer at 6000 r/min, and the PZT transducer was glued to the silicon wafer [see Fig. 3(e)]. The glue was cured in elevated temperature of 80 ◦ C for 24 h. After the integration of the silicon lens and the PZT transducer, another bulk-micromachined silicon wafer
CHOE et al.: PEPTIDE SYNTHESIS ON GLASS SUBSTRATE USING ACOUSTIC DROPLET EJECTOR
Fig. 4. Conceptual steps on how droplets of amino-acid are coupled onto the glass substrate: (a) free amine on the glass surface, (b) coupling of preactivated amino-acid to free amine on the glass, (c) capping of uncoupled free amines, (d) fmoc-deprotection for next mer coupling. (e1) coupling next amino acid. (e2) coupling FITC for optical measurement.
Fig. 5. spots.
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Synthesis of peptide ladders of nine different lengths over nine different
D. Synthesis of Peptide Ladder was added to act as a top cover providing a mechanism to maintain the liquid level constant. Then, the completed ejector was mounted on a dual-inline-package (DIP) socket [see Fig. 2(a)]. The dimension of the fabricated acoustic ejector is measured to be 6 mm (width) × 12 mm (length) × 1 mm (thickness). The ejector consists of ejecting chamber, liquid transfer channel, and liquid reservoir. The ejecting chamber, in which acoustic lens-with-air-reflector is sitting, occupies 3 mm (width) × 3 mm (length) × 1 mm (thickness). The dimensions of the liquid transfer channel and reservoir can easily be reduced or avoided by using other liquid supply techniques such as a tube and/or liquid cartridge. C. Coupling Amino-Acid Onto the Glass Surface To couple amino acids, 60 nL (corresponding to 300 droplets of 0.2 nL) of the preactivated solution (6.7 mM) of fmoc-amino acid with 6.7 mM of BOP, HOBt, and DIPEA in N-methyl2-pyrrolidone (NMP) was dispensed using an acoustic droplet ejector onto the amine-terminated spots on the surface modified glass slide, and let to react for 60 min at room temperature [see Fig. 4(b)]. The uncoupled free amine groups in the active spots were capped with 10% (v/v) solution of acetic anhydride and pyridine (1:1) in NMP for 30 min at room temperature [see Fig. 4(c)]. The fmoc-protection group was then removed with 20% (v/v) solution of diethylamine in NMP for 30 min at room temperature to liberate free-amine group [see Fig. 4(d)]. The liberated free-amine group was coupled with next mer aminoacid [see Fig. 4(e1)] or reacted with a 5.0 mM solution of FITC in NMP for 2 h at room temperature to attach FITC fluorescent tag at the end of the peptide for an optical measurement that quantifies the number of the coupled amino acid [see Fig. 4(e2)].
A peptide ladder consisting of glycine from 1-mer to 9-mers was synthesized on nine different spots on a surface modified glass slide (see Fig. 5). At the start of the synthesis, about 60 nL of preactivated amino-acid solution was dispensed onto all the nine active spots using the fabricated acoustic droplet ejector. The glass slide was placed about 2 mm right above the opening of the ejecting chamber (through which droplets are vertically ejected). After dispensing 60 nL of the solution on the desired spot, the glass slide was moved so that the ejector can dispense the solution onto the next active spot. Once the ejector dispenses the preactivated amino acid solution onto all of the nine active spots, the active spots were left for 60 min at room temperature for reaction with the dispensed amino acid to occur. After washing the whole glass slide two times with NMP and one time with methyl alcohol, we blow-dried the glass slide with dry nitrogen. The uncoupled free-amine groups in the active spots were capped for 30 min by dispensing 200 nL of capping solution with a pipette. Then, after washing out the capping solution, we removed fmoc-protection group from eight active spots (out of the nine total spots) by dispensing 200 nL fmocdeprotection solution, so that the eight spots would be ready to be coupled with next-mer amino acid. At this moment, the ninth spot was not active anymore, and it had only 1-mer-long glycine peptide to the end of the experiment. After washing the fmoc-deprotection solution, we moved the glass slide back onto the ejector for dispensing next mer amino acid. By repeating the steps above, all the nine spots had different lengths of peptide from 1 to 9 mers (see Fig. 5). When all the nine spots had desired number of peptides in the ladders, fmoc-protection groups of all the nine spots were removed so that FITC might be attached to the synthesized peptides (see Fig. 6). After FITC attachment, the light intensities
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Fig. 7. Experimental setup for ejecting droplets of preactivated amino-acid solution onto a surface-modified glass slide. Fig. 6. FITC attachment at the end of the peptide chains after the synthesis of the peptide ladders.
coming from the nine spots were measured with a fluorescent microscope at 532 nm. It took about 3 h to add 1-mer to a peptide chain with the technique used in the 9-mer peptide synthesis, mainly because the time demanding steps of washing, drying, reacting between chemicals. Also, preparing a bare glass slide to be ready for coupling with the first peptide mer took us about 2 days, because of cleaning any possible organic contamination, surface modification, washing in between surface modification steps, and drying in vacuum. However, with automation and optimization, the time can be reduced substantially. III. MEASUREMENT SETUP A. Droplet Ejection Setup The fabricated ejector was actuated with pulsed 17.3 MHz sinusoidal signals at 60 Hz pulse repetition frequency. The pulsed signal was amplified with an RF power amplifier to drive the ejector with a voltage amplitude of 160 Vp eak -to -p eak . A red LED was used as a light source to stroboscopically observe the ejection process through adjusting the delay between the device actuation and LED illumination (see Fig. 7). A chargecoupled device (CCD) camera being attached at the end of a long-working-range microscope was placed horizontally to record the ejection process to a computer. The glass slide with free-amine groups on its active spots was placed about 2 mm away from the ejector and aligned, so that the ejected droplets of preactivated amino-acid solution can ink the active spots of the glass slide. B. Light Intensity Measurement Setup A fluorescent microscope equipped with cooled CCD camera was used to detect the optical signals coming from FITC at 532 nm to confirm the synthesis. The cooled CCD camera captured images with exposure time of 30 s, and the captured images were transferred to a computer, and stored in lossless
image format (i.e., TIFF) to avoid data distortion. The light intensity was measured and quantified from the stored image using ImageJ software. IV. MEASUREMENT RESULTS A. Optimum Coupling Time Measurement The longer the coupling time, the better is the coupling efficiency (up to a certain time) in general. But the shorter the coupling time, the better is the throughput of the synthesis. To find out the optimal (or minimum) amino-acid coupling time in our experimental setup, 1-mer glycine was synthesized on a slide glass using a pipette with various reaction times (5, 10, 20, 30, 40, 60, and 90 min). All spots were reacted with FITC solution when the amino-acid coupling is finished. Since the light intensity coming from FITC labeled aminoacid represents the amount of the coupled amino-acid onto glass surface, the light intensity was measured as a function of the coupling time. The measured light intensities were normalized with the following equation, and plotted in Fig. 8 Inorm alized =
In I90 m in − Icapping
(1) only
where In , I90 m in , and Icapping only are the light intensities of the spots treated with n min coupling time, 90 min coupling time, and only with capping solution, respectively. As we can see in Fig. 8, the average light intensity of the spot with 60 min coupling time showed negligible difference from that of the spot with 90 min coupling time. Since the coupling did not improve when the reaction time was longer than 60 min, we fixed the amino-acid coupling time to 60 min in all the following experiments. B. Ejecting Condition Calibration The optimal driving condition for the droplet ejector such as the pulse width and actuation voltage depends on the liquid type. A 17.351 MHz pulsed sinusoidal signal with 2 μs pulse
CHOE et al.: PEPTIDE SYNTHESIS ON GLASS SUBSTRATE USING ACOUSTIC DROPLET EJECTOR
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Fig. 10. Photos showing the collected droplets of preactivated amino acid solution ejected by the acoustic ejector with silicon lens. Each liquid bump has 60 nL of the solution.
Fig. 8. Normalized light intensities measured from the amino-acid coupled spots as a function of coupling time.
Fig. 11. Photos of ejected droplets of the preactivated amino-acid solution that are on their way to a glass slide having free amines on its surface. Each photo shows droplet’s position at (a) 150 μs, (b) 450 μs, (c) 700 μs, and (d) 1000 μs after the droplet is ejected from the ejection chamber. Fig. 9. Photo of the droplet of preactivated amino acid solution just before being ejected out of the ejection chamber.
C. Light Intensity Measurement on Peptide Ladder
width was good to eject droplets of DI water. However, the pulse width had to be increased to 6 μs to eject droplets of preactivated amino acid in N-Methyl-2-Pyrrolidone (NMP) solution, since NMP has 1.7 times higher viscosity (1.7 × 10−3 Pa·s) than DI water (1.0 × 10−3 Pa·s). The droplet size of the acoustic ejector is primarily determined by the focal size which in turn depends on the wavelength of the acoustic wave produced from the transducer, and is very consistent for a given viscosity. In a low viscosity liquid such DI water and alcohols, the diameter of the ejected droplet is about the wavelength of the acoustic wave. With high viscosity liquid such as oil and NMP, the diameter of the ejected droplet is usually larger than the wavelength. However, the acoustic intensity level needed to eject droplets also is higher in case of liquid with higher viscosity, and is likely the reason for a larger droplet size. Even when the viscosity changes over a wide range (up to 55 cSt), the size of the ejected droplet varies less than 10%. The ejected droplets of NMP solution had 80 μm diameters as shown in Fig. 9, and we ejected 300 droplets to dispense 60 nL onto each spot on the glass slide (see Fig. 10)
Fluorescent light intensities of the nine spots of the peptide ladder were measured with a fluorescent microscope. Since the coupling efficiency is not 100%, the light intensity coming from the spot having a longer peptide ladder is lower. Comparing the measured light intensities on different spots, we can obtain the stepwise coupling efficiency or overall coupling efficiency. The preactivated amino-acid solution was dispensed onto the nine spots using acoustic ejector (see Fig. 11), and the fluorescent images taken from each spot of the ladder structure are shown in Fig. 12. The synthesis spot, where peptide is to be synthesized, is defined photolithographically, and the outside of the synthesis spot is perfluorinated to prevent the area from being coupled with peptide’s free-amine group. The perfluorinated surface is rough, and the rough surface makes it easy for fluorescent FITC to physically attach (nonspecifically) onto the area (i.e., the outside of the synthesis spot). Since the synthesis spot is well defined by precise photolithography technique, we can easily find where we should look at to measure the fluorescent light intensity solely due to selective binding between FITC and synthesized peptide. Fig. 13 shows monotonous decrease of the light intensity as the length of the synthesized peptide increased from 1-mer to
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Fig. 12. Fluorescent images of FITC-attached amino-acids that have been synthesized with the amino-acid droplets ejected by the acoustic ejector. Since the coupling efficiency is not 100%, the light intensity decreases as the length of the synthesized protein increases. Higher light intensity represents more population of the protein on the glass surface.
Fig. 13. Measured fluorescence light intensity versus the length of the synthesized protein. The intensity of the 8-mer protein is about 70% (=0.968 ) of that of the 1-mer protein, indicating a stepwise coupling efficiency of 96%.
9-mers if we exclude the light intensities measured from the spots having 2-mer and 3-mer long peptides. The light intensities coming from those spots were lower than the expected possibly because the quality of the free amine groups on those spots was not as good as the other locations. Since most of the spots showed a reasonable light intensity, the average stepwise coupling efficiency and overall coupling efficiency in synthesizing the 9-mer-long glycine peptide were calculated to be 96% and 70%, respectively. The synthesis results shown in Fig. 13 were from one experiment, though we carried out many repeated experiments to master and confirm the peptide synthesis protocol described in [12] both with the acoustic ejectors and conventional pipettes, as we focused on optimizing the acoustic ejector for synthesis of high-density peptide array. V. CONCLUSION This paper describes protein synthesis with various 200 pL peptides that are ejected onto a solid surface by MEMS droplet
ejectors. We have successfully synthesized 9-mers of glycine on modified glass surface using SPOT technique. Protein microarray can be fabricated through either immobilization of presynthesized protein chain or in situ solid-phase synthesis from peptides. SPOT technique is one of the in situ peptide synthesis methods that is cost effective, easy to automate, and has simple experimental procedure. But SPOT has traditionally relied on micropipettes for spotting active areas with peptides, and is limited in its scalability. The acoustic droplet ejector described in this paper, on the other, is scalable, and will allow massive parallel protein synthesis on a solid substrate from peptides. The acoustic microdroplet ejector with air cavities formed by micromachined silicon was fabricated to dispense droplets of 200 pL on demand, and was used to synthesize a 9-mer-long ladder of glycine on a modified glass surface with 96% stepwise coupling efficiency. Though the active spot size was 1 mm in diameter in this preliminary study, the spot size can easily be reduced to a much smaller diameter with a relatively minor optimization effort. REFERENCES [1] R. B. Merrifield, “Solid phase peptide synthesis—Part I. The synthesis of a tetrapeptide,” J. Amer. Chemical Soc., vol. 85, no. 14, pp. 2149–2154, Jul. 1963. [2] A. R. Mitchell, S. B. H. Kent, M. Engelhard, and R. B. Merrifield, “A new synthetic route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl)phenyl acetamidomethyl-resin, an improved support for solid-phase peptide synthesis,” J. Organic Chem., vol. 43, no. 14, pp. 2845–2852, Jul. 1978. [3] S. S. Wang, “p-Alkoxybenzyl alcohol resin and p-Alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments,” J. Amer. Chem. Soc., vol. 95, no. 4, pp. 1328–1333, Feb. 1973. [4] G. R. Matsueda and J. M. Stewart, “A p-Methylbenzhydrylamine resin for improved solid-phase synthesis of peptide amides,” Peptides, vol. 2, no. 1, pp. 45–50, Jan. 1981. [5] P. Sieber, “A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method,” Tetrahedron Lett., vol. 28, no. 19, pp. 2107–2111, Jan. 1987. [6] R. Frank, “Spot-synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support,” Tetrahedron, vol. 48, no. 42, pp. 9217–9232, 1992. [7] R. Frank, “The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—Principles and applications,” J. Immunological Methods, vol. 267, no. 1, pp. 13–26, Sep. 2002. [8] N. Heine, T. Ast, J. Schneider-Mergener, U. Reineke, L. Germeroth, and H. Wenschuh, “Synthesis and screening of peptoid arrays on cellulose membranes,” Tetrahedron, vol. 59, no. 50, pp. 9919–9930, Dec. 2003. [9] X. Espanel and R. H. Huijsduijnen, “Applying the SPOT peptide synthesis procedure to the study of protein tyrosine phosphatase substrate specificity: Probing for the heavenly match in vitro,” Methods, vol. 35, no. 1, pp. 64–72, Jan. 2005. [10] H. E. Blackwell, “Hitting the SPOT: Small-molecule macroarrays advance combinatorial synthesis,” Current Opinion Chem. Biol., vol. 10, no. 3, pp. 203–212, Jun. 2006. [11] K. Hilpert, D. F. H. Winkler, and R. E. W. Hancock, “Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion,” Nature Protocols, vol. 2, pp. 1333–1349, May 2007. [12] D. H. Kim, D. S. Shin, and Y. S. Lee, “Spot arrays on modified glass surface for efficient SPOT synthesis and on-chip bioassay of peptides,” J. Peptide Sci., vol. 13, no. 10, pp. 625–633, Oct. 2007. [13] C. Y. Lee, H. Yu, and E. S. Kim, “Acoustic ejector with novel lens employing air-reflectors,” in Proc. 19th IEEE Int. Conf. Micro Electro Mech. Syst., pp. 170–173, 2006.
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