Reactive inkjet printing and functional inks : a versatile route to
new programmed materials
Citation for published version (APA):
Delaney, J. T. (2010). Reactive inkjet printing and functional inks : a versatile route to new programmed materials. Technische Universiteit Eindhoven. https://doi.org/10.6100/IR685858
DOI:
10.6100/IR685858
Document status and date: Published: 01/01/2010 Document Version:
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1
INKS
:
A VERSATILE ROUTE TO NEW
PROGRAMMED MATERIALS
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Technische Universiteit Eindhoven, op gezag van de rector magnificus, prof.dr.ir. C.J. van Duijn, voor een
commissie aangewezen door het College voor Promoties in het openbaar te verdedigen
op dinsdag 7 september 2010 om 16.00 uur
door
Joseph Thomas Delaney, Jr.
2 Dit proefschrift is goedgekeurd door de promotor: prof.dr. U.S. Schubert
The research described in this thesis forms part of the research program of the Dutch Polymer Institute (DPI, P.O. Box 902, 5600 MB, Eindhoven), Technology Area High Throughput Experimentation, DPI project #502.
A catalogue record is available from the Eindhoven University of Technology Library Proefschrift, ISBN: 978-90-386-2325-2
Cover design by Joseph T. Delaney, Jr.
Printed at: Ipskamp Drukkers B.V., Enschede, The Netherlands
3
Reactive inkjet printing & functional inks: a versatile route to
new programmed materials
Kerncommissie:
Prof. Dr. Ulrich S. Schubert (Eindhoven University of Technology)
Prof. Mohan Edirisinghe (University College of London)
Prof. Jean-François Gohy (Eindhoven University of Technology) Prof. Dr. Dick J. Broer (Eindhoven University of Technology) Prof. Dr. Ir. René Janssen (Eindhoven University of Technology) Overige Commissieleden:
Prof. Dr. Patrick J. Smith (University of Sheffield) Dr. Jolke Perelaer (Friedrich-Schiller-University of Jena)
4 “Be a scientist, and save the world.”
-Markus Antonietti, 2010
“In the natural sciences these are and have been, and are most surely likely to continue to be heroic days.”
-J. Robert Oppenheimer, in ‘Prospects in the Arts and Sciences’, Bulletin of the
5
1. An introduction to reactive inkjet printing: inkjet printing of proteins ... 7
1. 1 Introduction ... 8
1. 2 Developments in biological high-throughput experimentation ... 9
1. 3 Biosensors, and immunoassay tests ... 10
1. 4 Proteomics ... 18
1. 5 Peptide synthesis ... 20
1. 6 Inkjet printing, proteins, and X-ray crystallography ... 21
1. 7 Limitations to protein printing ... 23
1. 8 Conclusion and outlook ... 27
1. 9 Outline of the thesis ... 29
1. 10 References ... 30
2. Inkjet printing of hydrogel porogens ... 37
2.1 Introduction ... 38
2.2 Results and discussion ... 41
2.3 Summary ... 44
2.4 Experimental section ... 46
2.5 References ... 50
3. Organ weaving – a new strategy for artificial organ development ... 53
3.1 Introduction ... 54
3.2 Results and discussion ... 57
3.3 Summary and outlook ... 63
3.4 Experimental section ... 65
3.5 References ... 70
4. ‘One cell-one well’ — a new approach to inkjet printing single cell microarrays ... 75
4.1 Introduction ... 76
4.2 Results and discussion ... 78
4.3 Summary and outlook ... 84
4.4 Experimental section ... 86
6
5. Inkjet goes ‘click’— preparation and functionalization of self-assembled monolayers using
reactive inkjet printing ... 95
5.1 Introduction ... 96
5.2 Results and discussion ... 98
5.3 Summary and outlook ... 104
5.4 Experimental section ... 105
5.5 References ... 107
6. Reactive inkjet printing of irreversibly crosslinked networks ... 111
6.1 Introduction ... 112
6.2 Reactive inkjet printing of polyurethanes ... 112
6.2.1. Introduction ... 112
6.2.2. Results and discussion ... 114
6.2.3. Summary and outlook ... 118
6.2.4. Experimental section ... 119
6.3 Reactive printing of ionogels ... 120
6.3.1. Introduction ... 120
6.3.2. Results and discussion ... 122
6.3.3. Summary and outlook ... 132
6.3.4. Experimental section ... 133
6.4 Conclusions ... 134
6.5 References ... 135
Thesis summary ... 139
Appendix: Theoretical examination of the multi-pass single particulate experiment design ... 146
Curriculum Vitae ... 151
List of publications ... 152
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An introduction to reactive inkjet printing: inkjet printing of proteins
Abstract:
This chapter is intended to provide an introduction to the field of inkjet printing. Using a review of the current status of the field of inkjet technology with protein-related applications as an illustrative example, the goal is to provide a brief history of inkjet printing, as well as a discussion of the advantages and challenge of employing the technology with proteins, using a number of selected applications as examples. The chapter then continues with a view of future research directions. As a category of printable, reactive functional materials, a survey of reactive protein printing is particularly illustrative of the variety of issues and applications afforded by inkjet technology. The chapter concludes with an outline of the thesis, and the general layout of the chapters.
Parts of this chapter have been published: J. T. Delaney, P. J. Smith, U. S. Schubert, Soft Matter
8
1. 1 Introduction
The aim of this chapter is to provide a brief introduction to inkjet printing, including a general description of some of the highlights of reactive inkjet printing, as illustrated by a survey of the current state of the art of inkjet printing of proteins. Reactive inkjet printing, defined herein as the handling of reactive fluids using inkjet technology, encompasses a broad range of materials science research activities. Due to the intrinsically vast variety found in proteomes, the processing sensitivity of active protein structures, as well as the significant extension of the reach of instrumental analysis techniques in recent decades, disciplines such as proteomics and applied protein chemistry have made particularly intensive use of microfluidics tools such as inkjet printing; the net result is that a study of the combination of how inkjet technology is applied to proteins serves as a particularly effective exercise in describing the general scope of reactive inkjet technology as it stands today. Finally, the conclusion of this chapter will include some summary statements on the state-of-the-art in inkjet printing of proteins, as well as provide a general discussion of the outline of the subsequent chapters of this thesis.
Inkjet printing is a versatile technique that has been widely used for the direct-writing of two-dimensional features, and, less widely but increasingly, for three-two-dimensional structures. The mild conditions afforded by inkjet printing make it particularly suited for handling biological samples since contamination can be minimized and waste is reduced. Another attraction is the accurate placement of pre-determined quantities of material, which can be performed without the need for previous patterning steps. Inkjet printing describes the use of electrical actuators to eject picoliter volumes of liquid from micron-sized apertures onto a substrate in a defined pattern. It has gained wide acceptance in industry as a tool for basic discovery and as a proven means of rapid fabrication. While the most popularly identifiable application of inkjet technology remains that of printing paper documents, it has also been used in fields as diverse as organic electronics, chemical synthesis, sensor fabrication, combinatorial chemistry and biology.[1-3]
The term ‘direct-write’ describes fabrication methods that employ a computer-controlled translation stage, which either moves a pattern-generating device (such as an inkjet nozzle) or the substrate with the purpose of creating ‘prints’ with controlled architecture and composition.[4] Inkjet printing can be divided into two broad categories: drop-on-demand or
9
which droplets are deflected from a continuous stream to a substrate when needed. Inkjet can be further subdivided according to the specific means of generating droplets, such as piezoelectric, thermal and electrostatic. As expected, each of these particular techniques has specific strengths, and limitations in terms of their applicability to bio-printing. Such variables include the operating temperature range, material throughput, reproducibility of droplets, precision of deposition, range of printable viscosities, range of shear forces within the nozzle, reservoir volume and the number of fluids that may be printed during a single experiment. There is a lot of overlap in terms of applicability for these different techniques since many bio-printing applications involve the deposition of dilute solutions/suspensions under near-ambient conditions with spatial resolution requirements measured in the order of tens of microns. A number of publications have covered the topic of comparative instrument performance reasonably well in recent years.[5-8]
Proteins are macromolecules that are made up of amino acids, which have been arranged in a linear chain, and are joined together by peptide bonds between the carboxyl and amino groups. Proteins are essential parts of organisms and participate in a large number of processes within cells. The use of inkjet printing to deposit proteins is appealing since many proteins, such as expensive, carefully purified enzymes, or biomarkers from biopsies, are only available in minute, finite quantities, and must be handled using the most materially efficient techniques available. Many protein-based devices, such as biochips based on active enzymes, immunoglobulins or biomarkers, require multi-micron resolution of spatially arranged arrays of different materials. A technique that can reproducibly dispense such components under mild conditions is understandably useful.
1. 2 Developments in biological highthroughput experimentation
High-throughput experimentation (HTE) refers to a general experimental methodology whereby large libraries or arrays of materials are prepared, and analysed. A famous early example of high-throughput screening was Thomas A. Edison’s development of light-bulb materials,[9] wherein approximately 6,000 different filament materials were screened. The advantages to HTE are that large numbers of compounds and materials may be efficiently and systematically studied, which allows researchers to empirically answer many scientific
10
questions that would otherwise be intractable using more manual, sequential approaches.[10] Arguably, no other branch of science has embraced the high-throughput philosophy more than modern life sciences, and HTE involving proteins exemplifies this trend.
While inkjet printing has been around for several decades, the field of bio-printing only began to grow in the mid-90’s, with a rapid increase after 2000. There are several factors that could account for this increase: it was only in 1985 that inkjet printers began to be used for dispensing functional materials, other than inks, in text and graphics.[11] By 1985, sensors were already being fabricated in a multi-analyte array format,[12] which could simultaneously measure concentrations of glucose, and urea. Arrayed multi-analyte immunoassays followed soon after,[13] demonstrating the strong interest in spatially-arrayed biosystems. By 1987, the
first patent for an enzyme-based biosensor prepared by inkjet printing had been applied for, and was granted the following year.[14]
Perhaps another reason for the increased use of inkjet as a manufacturing technique could be the advent of software such as Windows 95, which made the technique more accessible. This inexpensive, easy-to-use, almost ubiquitous software enabled non-computer experts the ability to control ink deposition onto substrates in organized patterns. Another reason for this sudden increase in interest can be traced to the affordability, and availability of inexpensive office inkjet printers, which researchers were able to modify to meet their needs.
1. 3 Biosensors, and immunoassay tests
One of the challenges of studying and working with purified proteins is the limited availability of many of them. While otherwise insufficient quantities of DNA or RNA samples may be readily amplified using techniques like the polymerase chain reaction (i.e. PCR), there is no equivalent technique available for copying proteins.[15] In many forensic applications, where a given supply of starting material may be finite, the experimenter must plan tests carefully to allow for basic identification, and characterization of the proteins, and may not have any remaining material left over as a reserve for any subsequent testing. In other cases, the process of obtaining, and purifying proteins may be especially costly or laborious. In these instances, it is important that such materials be handled as parsimoniously
11
as possible. Inkjet printing, particularly drop-on-demand techniques, addresses many of these issues,[16] by using pico- to nanoliter-sized quantities of buffered protein solutions which may be precisely dispensed to a designated sample space, with minimal waste.
From a life sciences/microelectromechanical systems (MEMS) standpoint, the ability to array proteins with good precision in uniform quantities is a useful feature both for the preparation of analytical samples, as well as for the fabrication of devices. Protein microarrays allow for the study of specific interactions between immobilized individual proteins, and other biological systems.[17] This ability translates directly into applications such as point-of-care clinical diagnostics, and biosensors.
One area of increasing research has been in the field of biosensors,[18] and/or biologically active MEMS (bioMEMS).[18-19] A biosensor is an analytical device that uses antibodies, enzymes, nucleic acids, micro-organisms, isolated cells, or other biologically derived systems as a sensing element.[20-22] Some of the earliest examples of these include an enzyme-based ion-sensitive field effect transistor (ISFET),[23] which may be considered as the first example of bioMEMS. In the most recent devices of third generation, the sensing element is directly bound to the electronic system that transduces, and amplifies the signal.[24-25]
There are several unique advantages to bioMEMS over other analytical tools for measuring certain trace compounds.[26] In terms of cost, very small quantities of material are required to
make sensors, and inkjet deposition lends itself well to mass production, allowing for sensors in many applications to be treated as disposable. The inherently small size of the detectors involved implies the need for only small quantities of material for analysis, resulting in greater sensitivity.[27] Also, the need for a smaller amount of analyte offers faster detector response times, since less time is required for the substrate to interact with the analyte before a signal is detected. The use of enzymes, and antibodies as transducers also results in unmatchable specificity for bioactive compounds.
The limited stability of many biological activity materials involved constrain the number of techniques that can be used to fabricate bioMEMS. Techniques such as vapor deposition and photolithography are not well-suited to handling bioactive systems such as enzymes and
12
microorganisms. Inkjet printing, on the other hand, typically involves highly-reproducible, non-contact deposition of fluids under mild conditions, and therefore lends itself well to bioMEMS fabrication. The droplet volumes involved, typically ranging from 2 pL to 5 nL at a rate of 0 to 25 kHz for drop-on-demand printers (and up to 1 MHz for continuous printheads)[18] are especially useful for high-volume fabrication.
One of the areas where inkjet printed biosensors have gained the most interest from a commercial standpoint has been disposable, point-of-care diagnostic products, such as glucose sensors.[28-32] While thick film biosensors have been manufactured using screen printing,[33-35] the need for smaller quantities of enzyme, high reproducibility, and general minimization of waste[31] makes inkjet an attractive alternative. The first report in the open
literature of an inkjet-fabricated glucose sensor occurred in 1988, making it one of the earliest examples of inkjet deposition of functional materials.[36] Since the enzyme-catalyzed redox reaction of glucose results in an easily measurable amperometric signal, the process is well-suited for the application of a printable biosensor. In a typical example,[28] an aqueous solution of the enzyme glucose oxidase (GOD) is printed with a conductive polymer such as poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS) into a conductive layer of ITO, which is then encapsulated by a semi-permeable membrane of cellulose acetate (Figure 1.1).
Many amperometric, and potentiometric enzymes that have been screen printed into working biosensors have been subsequently manufactured using inkjet technology. Biosensors designed to measure the total protein content of liquid samples[37] have been developed in this fashion.[30] Likewise, biosensors for the quantitation of L-lactate (an industrially important measurement for the dairy industry) was reported to be made by screen printing,[38]
followed shortly afterwards by inkjet printing.[39] Clinically ubiquitous amperometric enzymes such as horseradish peroxidase follow this same trend: horseradish peroxidase-based biosensors were first screen printed ca. 1987,[40-41] and were then printed by inkjet six years later,[42] with new applications continuing to be developed on a regular basis.[43-45]
13
Figure 1.1. Inkjet printing of a glucose biosensor prototype.[28]
Another recent application of printed amperometric biosensors involves the detection of viable bacterial pathogens using a combination of bacteriophages, and redox enzymes. First developed for Escherichia coli,[46] this strategy has since been recently extended to the
detection of extremely low concentrations (~10 colony-forming units per mL) of Bacillus
anthracis (the anthrax pathogen), and Mycobacterium tuberculosis (the tuberculosis
pathogen).[47] In this scheme, the bacterial suspension is exposed to the biosensor, which is loaded with a pathogen-specific phage (a type of virus that only infects a specific type of bacterium); if the bacteria of interest are present, the phage will infect them, causing the cells to undergo lysis, and release their contents, including sugars; as this happens, the biosensor records this using a β-galactosidase electrochemical assay. Due to the inherent specificity of bacteriophages, the method is not only specific to a given pathogen, but is also specific to viable bacteria only, which makes this approach even more specific than analyses such as PCR. In addition, this entire process takes place within 8 h, which represents a considerable time savings compared to culturing methodologies (24 to 48 h). Such sensors are inexpensive, easily mass produced, operable under field conditions, are not affected by the presence of other microorganisms, and do not require early preparation of the samples. While the earliest applications of this phage/enzyme biosensor system have been related to three species of bacteria associated with water pollutants, and biowarfare agents, it is easy to
14
envision a wide array of public health, environmental, and medical applications for such devices.[48]
Perhaps the most common application of biosensors are immunoassays,[49] including enzyme-linked immunosorbant assay (ELISA), and related antigen-antibody-complex based techniques. Immunoassays involves the measure of the level of a particular substance in a sample using the reaction of an antibody to its corresponding antigen. Depending on the design, and application, they can provide both qualitative, and quantitative analysis of cytokines, growth factors, antibodies, food allergens, and other biomarkers for brain trauma, heart disease, pathogens, cancer, blood type, and pregnancy. Originally conceived in the 1960’s, the use of antibodies on solid supports was a technology that lent itself well to the use of multianalyte microarrays,[50] and consequently print-based manufacturing, and processing techniques. There are a numerous variations on this theme, with several excellent reviews covering the different forms of this system, along with their strengths, and limitations.[51-52] Outlining all the different available types of immunosorbent assays is beyond the scope of this overview, though for the sake of illustration, it is worth examining one general category, the sandwich-type immunoassay, in more detail (Figure 1.2). In this assay, the surface of a titer plate is coated with a known quantity of capture antibody for a particular antigen of interest. The analytical sample containing the antigen of interest is then applied to the plate, and the antigen binds to the antibody. The plate is then washed to remove any unbound antigen, and a detecting antibody is added. The plate is washed again to remove any unbound detecting antibody, and is then treated with an enzyme-linked secondary antibody, which binds to the detecting antibody. The plate is washed again, and finally, a chemical reagent is added that is converted by the enzyme into a fluorescent material. If the analyte is present, the sample should yield a fluorescent signal at the end of the process.
15 Analyte Analyte An alyte Analy te Substr. Substr.
Figure 1.2. General scheme for one example of a sandwich-type immunoassay.
Recently, an automated version of a sandwich-type immunoassay was developed by James B. Delehanty, and Frances S. Ligler.[53] The substrate of capture antibodies was prepared using
piezoelectric inkjet printing, allowing for an entire microarray to be employed in a single test. The result is that a single sample can be quickly screened in the field for a diverse number of relevant potential pathogens. In their first reported application for this instrument, the device was able to simultaneously detect both proteins, and bacteria – a first for a single immunoassay test.[53] Specifically, the authors were able to detect cholera toxin,
staphylococcal enterotoxin B (a protein normally associated with food poison), ricin, and
Bacillus globigii (a commonly-used substitute for the more biohazardous B. anthracis). A
field-ready prototype developed by the U.S. Naval Research Laboratory[54] of a biodefense sensor system based on this technique was published in 2003, which also demonstrated its efficacy at detecting the pathogen Salmonella in food samples, as well (Figure 1.3).
16
Figure 1.3. Prototype of a portable array biosensor.[54]
In terms of current, commercialized applications of bio-printing, inkjet printed proteins have already been used for almost two decades in the form of immunoassay tests,[55] including point-of-care diagnostics for determining blood types, and pregnancy status. One of the earliest examples of this was the use of inkjet to manufacture a four antibody test to measure blood types (Figure 1.4).[18, 55] In this application, four different blood typing reagents are printed onto a nitrocellulose substrate in the form of the characters A, B, and +, with the “+” made up of the control-, and RH antibodies. In the presence of the appropriate antigen, the test is designed to visibly display the corresponding symbol. A similar +/- antibody readable display scheme based on the antibody response to the β-subunit of human chorionic gonadotropin (β-hCG) is used in common over-the-counter pregnancy tests.[56]
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While not fully commercialized yet, one of the most interesting applications of inkjet towards immunoassay diagnostics is the detection, and analysis of biomarkers, particularly for studying fetal development, and detecting or monitoring different types of cancer cells.[57] In a recent example,[58] a biochip was developed to simultaneously detect, and quantify urokinase type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor (VEGF) in extracts of breast cancer tissues (taken from core biopsies). As more biomarkers for cancers are identified, the hope is that techniques such as these will not only be able to identify the presence of cancers, but also provide details on the specific active biological processes taking place in a given sample, allowing for more tailored treatment.
Another new biosensor-related application of inkjet has been towards the development of prosthetic retinas.[59] In this embodiment, inkjet dispensers (from a recycled Epson 740 Stylus color printer) are used to deliver a solution of the neurotransmitter bradykinin to stimulate living rat pheochromocytoma cells. Unlike other inkjet biosensor applications, inkjet is an integral part of the device, rather than the means of fabrication. The current working example was intended strictly as a proof-of-concept exercise to demonstrate the feasibility of inkjet as a biological interface; the long-term goal is to create a multiple stimulation actuator device that could be used to release neurotransmitters in a patterned array, to act as a sort of image actuator (Figure 1.5).
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1. 4 Proteomics
Proteomics, the large-scale study of the function, and structure of proteins, poses some unique technical challenges not found in other branches of bioinformatics, such as genomics. While the genome for any given organism is constant and uniform in most healthy cells throughout an organism’s lifetime, the proteome varies from cell to cell and is constantly changing as the cells interact with the genome and surrounding environment. This high degree of variability make proteomics a uniquely challenging field, and at the same time, offers unique promises in understanding biological processes.
In general, inkjet printing has been employed in proteomics as a high-throughput tool for the preparation of samples for analysis (e.g. printing protein arrays[60-63]) as well as for peptide synthesis. In the former category, inkjet is used as an arrayer[16] to study the proteome[64] of a
given biological system’s endogenous peptides, or derivatives thereof. In the latter category, inkjet is used as a dispensing technique for reagents for the de novo synthesis of peptides. Several examples of both categories of inkjet-assisted applications to proteomics (inkjet printing biologically derived proteomes, versus using inkjet as a tool for arrayed de novo peptide synthesis) exist in the literature.
The most commonly reported inkjet-assisted proteome analyses involve matrix-assisted laser desorption ionization (MALDI)-based mass spectroscopic techniques, including MALDI coupled with quadrupole ion trap mass spectrometry (MALDI-QIT-TOF),[65-66] MALDI
coupled with time-of-flight mass spectrometry (MALDI-TOF-MS),[67-70] and surface-enhanced laser desorption-ionization mass spectrometry (SELDI-MS).[71] It is worth also mentioning that purely non-destructive vibrational spetroscopic techniques such as Raman spectroscopy have also been reported as well,[72] but are generally not as popular for characterizing peptide arrays. In terms of proteome analysis, inkjet printing applications can be further divided in two subgroups: inkjet deposition of proteins (or other macromolecules[71, 73-74]) onto an analytical substrate, and inkjet deposition of reagents, and matrix materials onto proteins already immobilized on a substrate.
One of the more interesting recent applications of inkjet-assisted protein analysis has been MALDI imaging.[65-66, 75-76] In MALDI imaging, proteins that are already immobilized on a
19
surface (e.g. Western blot membrane, electrophoresis gel,[77] lectin blot,[68] or tissue
cross-sections[65-66, 75-76, 78]) are treated with matrix material, and sometimes a protease such as trypsin to prepare the sample for analysis. The surface is then scanned using MALDI-MS, and the data analyzed. For MALDI imaging of tissues, electrophoresis gels, and blotting samples all require uniform, controlled dispensing of matrix material under conditions that minimize significant lateral migration of proteins on the surface, and inkjet is a method of choice for doing this.
As a tool for extending the capability of blotting techniques, the use of inkjet to apply matrix materials for MALDI experiments allows for a more direct primary structure analysis of the proteins (and post-translational modified protein derivatives) that make up the proteome being studied.[79] Prior to direct mass spectrometry of separated proteins from the gel, the process involved an additional extraction/separation step from the gel, followed by digestion of individual fractions.[80-81] By enabling mass spectrometry of the proteins directly from the electrophoresis gel, the analytical workflow becomes much more simplified. One recent example of tandem two-dimensional electrophoresis/MADLI-QIT-TOF analysis using inkjet involved the study of post-translation phosphorylated proteins.[77] In this example, phosphorylproteins from A-431 human epidermoid carcinoma cells, which were detected using anti-phosphotyrosine antibody, and stained using Direct Blue 71 (Figure 1.6). On-membrane digestion was carried out by dispensing trypsin and MALDI matrix material using inkjet into specific patterns onto the surface, which allowed for the improved on-membrane protein mass fingerprint analysis of protein spots without the challenge of cross-contamination of proximate proteins. By exposing a second sample of the same line of carcinoma cells to epidermal growth factor (EGF), they were able to study the differences in protein expression. As a result, the authors were able to detect several known proteins that are phosphorylated in an EGF-dependent manner.
20 f
Figure 1.6. Two-dimensional electrophoretic (2-DE) blot image of phospho-enriched whole cell lysates from
A-431/A-431 + EGF cell lysates. (a) Image visualized with Direct Blue 71 staining; (b) fluorescent image detected with an anti-phosphotyrosine antibody.[77]
1. 5 Peptide synthesis
The solid supported spot synthesis of libraries of peptides was reported some years ago.[82] While the number of naturally occurring amino acids is finite, implementing an inkjet system that could handle twenty amino acids plus additional reagents and solvents presents a technical challenge,[18] although efforts have been made in recent years to produce dedicated
21
One such effort uses inkjet printing to dispense amino acid reagents for solid-phase peptide synthesis onto the surface of a compact disc.[70] Using this scheme, up to 2,500 spots were prepared, employing up to 24 different reagents, in roughly three minutes (Figure 1.7). One of the unique features of this configuration is that the centrifugal force on the spinning surface may be utilized in washing steps to remove unreacted reagents prior to subsequent synthetic steps. Once the reaction sequence has been completed, the disc can be transferred for analysis by MALDI-TOF. This instrument was custom designed, though the proof-of-concept demonstrates that it could be readily applied to peptide, and nucleotide arrays using the popular ‘spot synthesis’ technique.[82]
Figure 1.7. Top: Bio Disc Synthesizer distribution principle. (1) CD type support, (2) disc zero position, (3) rotary
angle of support, (4) inkjet valve holder block, (5) inkjet integrated valve/nozzle combination, (6) swing arm, and ‘r’ is the rotary angle of the swing arm. Bottom: array of 2500 spots on the solid phase disc surface.[70]
1. 6 Inkjet printing, proteins, and Xray crystallography
To fully understand the function of a protein at the atomic level requires an understanding of its three-dimensional structure. While DNA sequencing has enabled the prediction of the
22
primary structure of proteins, the functional structures of proteins are not singularly defined as a result of one-dimensional strings of letters, but by how these chains of monomers fold into three-dimensional objects. Presently, in terms of characterizing secondary, and tertiary structures of proteins, by far the most widely employed technique currently used is single-crystal X-ray single-crystallography, with several examples in the literature demonstrating the usefulness of inkjet in preparing analytical samples.[61, 84-86] One of the challenges of this approach is growing single crystals – a process that is still not well understood.[87-89] Despite tremendous achievements in recent years made by this field, protein crystallogenesis is still primarily a trial-and-error art rather than a rigorously defined science: variations in pH values, precipitating reagents, temperature, cations/anions, and ligands can all be varied independently for the purpose of generating non-denatured protein crystals. To vary these parameters in an efficient workflow implies parallel experiment design. Since purified bioactive proteins are frequently only available in microgram quantities, the need for economy in discovering the right crystal growing conditions means that small, precise quantities of solution (at near-saturated concentrations) must be used. For these reasons, inkjet is well-suited as a tool for performing this task.
One recent approach to this has been the use of inkjet printing of proteins in a “hanging drop” vapor diffusion method for studying protein crystallogenesis.[84] In this method, a drop containing protein, reagent, and precipitating agent, at concentrations where the protein is almost saturated, is equilibrated against a reservoir containing a supersaturated precipitating agent buffer solution. Vapor diffusion between the protein solution droplet, and the reservoir causes the concentration in the droplet to increase, supersaturate, and ultimately crystallize.[84, 90] For each spot in a 96 array, a small aliquot (approximately 2 μL) of near-saturated protein solution was deposited, along with an additional aliquot of the 96 different stock solutions (Figure 1.8). With further refinements, the scale was reduced, from 2 to 4 μL of droplet volume per sample to 250 nL. After allowing the protein in the droplets the opportunity to crystallize over time (18 days), the samples are evaluated to see which conditions yielded useable crystals.
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Figure 1.8. Representation of automated “hanging drop” crystallization, and lysozyme crystals grown in a 4 μL
droplet, illustrating the basic steps: A) aliquots of protein-loaded solutions are inkjet-printed onto a glass slide in standard 96-well Society for Biomolecular Screening (SBS) format, which are then placed over wells of corresponding solvent mixtures, and allowed to develop, B, C) examples of protein crystals (lysozyme) prepared under different conditions.[84]
1. 7 Limitations to protein printing
While inkjet printing of proteins has been employed successfully with a wide array of materials, it is not without its own caveats for bio-printing.[91-93] In work by Okamoto, and coworkers, the sheer stress of inkjet on DNA was studied, and not found to be of much concern,[94] while Delehanty and Ligler examined the possibility of non-specific protein adsorption on the inner surfaces of the printer, and overcame this problem with the use of a sacrificial additive of bovine serum albumin (BSA).[86, 95] This phenomenon of non-specific
24
proteins, but is still something that should be considered when designing experiments – particularly when working with small sample volumes.
Prior to dealing with any protein-specific considerations, the first and foremost limitation to drop-on-demand inkjet printing of any material is whether or not the material can be formulated into an ink that is printable – i.e., that stable, repeatable droplets are able to be ejected from the nozzle, with uniform velocities and volumes. Generally speaking, the most important intrinsic physical properties determining printability of a given ink are viscosity η, density ρ, and surface tension γ, as well as the nozzle diameter of the printing system, a.[96] Using a modified form of the Navier-Stokes equation, originally proposed by Fromm,[97] a dimensionless number Z based on the aforementioned physical properties can be used to estimate whether or not the right balance between the capillary force, inertial force, and viscous force will be achieved for stable droplet formation by the following equation:
12a Z
The mathematics involved in describing the theoretical printability of fluids based on their calculated Z number has been outlined elsewhere in greater detail by other authors,[96-99] and
in this chapter, it is sufficient to say that a solution is theoretically printable when 1 < Z < 10.[100] In most instances, any protein solution under consideration for printing will be dilute and aqueous, and thus have a density that is already predetermined; for a given nozzle, the printability of a given protein solution is determined by solution surface tension and viscosity. In the case of very dilute concentrations, the use of viscosity modifiers to increase the viscosity of a solution to improve printability has been reported,[93] but it is also very easy with proteins to obtain solutions where viscosity inhibits printability.
Proteins are by nature macromolecules, and consequently the viscosity of their solutions is often dramatically affected by changes in concentration. At higher concetrations, the capillary force is insufficient to break the fillament of the droplet during the ejection, and the droplet retracts back into the nozzle.[101] For polymers, the microrheological explanation for this behavior is that the coiled and folded polymer chains are elongated in the direction of flow into a stretched state, which is accompanied by a strong increase of the hydrodynamic
25
drag. However, most proteins, unlike synthetic linear polymers, are not randomly coiled chains; rather, most proteins tend to be carefully folded organized structures in their native state, and the degree to which proteins are either globular or fibrous plays an important role in their intrinsic viscosity, and consequently, determining the maximum concentration of a printable solution. What this means is that enzymes, messenger-, and transport proteins, which have a globular geometry, can be printed in solutions with higher concentrations than fibrous, so-called “scleroproteins” such as keratins, collagens, elastins. This viscoelasticity has a significant impact on the concentration limits of printable solutions of specific categories of important proteins. Globular proteins such as BSA may be easily printable in concentrations of 10 wt-% or more with common dampened nozzles, but collagen(I) solutions in concentrations of 0.3-0.5 wt-% (a range commonly used for tissue engineering applications), are unprintable with the same devices. While a common technqiue for improving the printability of viscous inks is to raise the printing temperature, there are practical limits with proteins that mean that this can only be done to a very limited degree. The result of this is that many proteins used as scaffold materials for artificial tissues are not readily dispensible in the concentrations otherwise commonly applied.
In addition to protein adsorption, another possible experimental error exists in inkjet bio-printing of proteins: in a recent publication by Nishioka et al.,[92] the authors studied the biological activity of the model enzyme peroxidase after printing. As Derby pointed out in his recent publication on bioprinting,[100] drop-on-demand printing processes involve shear rates in the range of 2×104 to 2×106s-1; while such shear rates pose no foreseeable problems for small, globular proteins,[102] they are sufficiently high to anticipate problems with maintaining the structural integrity of some of their more fragile, larger, counterparts.[103-106] According to the account offered by Nishioka et al.,[92] the higher compression rates used to
generate droplets caused more of the protein to denature and become biologically inactive; in the absence of stabilizing additives, they noted that reduced enzyme activity was observed under all printing conditions. By adding sugars (trehalose and/or glucose) to help preserve the enzymatic activity, the same group of authors found that they could reduce the effect of printing. This phenomenon has subsequently been studied by other research groups working on proteins in microfluidics.[93, 107-109] The precise mechanism by which this enzyme is
26
denatured through this process is still not well understood, and the extent to which this effect is typical of proteins in general is still not well-known (it is also worth noting that the same effect of using a sugar to preserve biological activity has also been employed for handling DNA plasmids to improve transfection efficiency post-printing[110]). As the demand for printing enzymes, and other proteins increases in the future, further research into this field will be required.
As a sort of corollary to the challenges of maintaining bioactivity of proteins while growing crystals from them, the fact that compression force alone can denature proteins raises some interesting questions on the mechanics taking place in proteins on the nano-scale. While Anfinsen's dogma may hold for small globular proteins, i.e. that the native secondary structure for a protein is determined primarily by the amino acid sequence, for more complex proteins with deeply folded pockets that act as sites for catalytic activity (such as most active enzymes),[111] this rule does not always hold true. In the case of post-translationally-modified proteins such as phosphoroproteins, metalloproteins, glycoproteins, not to mention proteins consisting of multiple subunits, the need for crystallography is that much greater. In understanding the relationship between mechanical stress and protein misfolding, it is highly desirable to have a controllable microrheological tool for studying the relationship between shear forces and subsequent bioactivity. Techniques such as single-molecule force microscopy have gained some popularity in recent years,[112-115] but such experiments are often challenging to set-up, and in many cases, a simpler, more statistical analysis of a “bulk” sample of proteins in solution may be sufficient, or in fact more desirable. Jaspe and Hagen reported experiments on the sheer rate of a protein solution passed through a capillary of a similar size to that of a standard inkjet nozzle to evaluate the effect of shear stress on protein misfolding.[102] By extension, the use of an inkjet capillary nozzle would allow such experiments to be undertaken where the sheer stress could be systematically varied by adjusting the voltage and pulse width, and the resulting droplets could be collected separately to quantify the precise relationship between shear rates and the threshold for inelastic misfolding.[113] Since droplets at each given shear rate can be deposited into a spatially adressable array, the direct write feature enables a “permanent record” of the misfolding threshold to be made.
27
An understanding of the relationship between the limitations of protein mechanical stability have a wide range of immediate real-world applications, from understanding the biophysics of pathology-related proteins, to formulating protein-derrived therapeutics,[116] to understanding numerous misfolding mathologies. Perhaps a more in-depth study of this phenomenon may help to illustrate some aspects of nano-mechanics of proteins, with an ancillary benefit of understanding how to handle proteins more effectively.
1. 8 Conclusion and outlook
The inkjet printing of biomaterials has been readily adopted by a wide number of research groups and commercial concerns around the world; there are several reasons for this. Inkjet printing is fairly straightforward to use and reasonably priced when compared to other additive microfabrication techniques. One of its strengths is that it accurately dispenses small amounts of liquid, typically picoliters. These low volumes allow researchers to sparingly use valuable proteins, such as purified enzymes, and place them in the desired location. Inkjet printing is also a non-impact printing technique; droplets are ejected from a nozzle and land upon the substrate. The non-impact nature of inkjet printing aids in the reduction of contamination when dealing with purified materials. Another strength of inkjet printing is that it is a ‘maskless’ patterning technology. The same power that has led to the technique being used in millions of offices and homes can also be used in protein-based research. The material can be precisely positioned without recourse to a number of earlier patterning steps. This reduction in the number of processing steps makes the technique cheaper and more importantly reduces waste. Many earlier biosensors and immunoassays were prepared by screen-printing (as discussed earlier), which meant important and sensitive materials were wasted due to them being coated on the screen. Screen-printing also requires that one physically swap between screens, which either need to be cleaned and stored or generated anew, whereas with inkjet printing the user switches between computer programs.
The ability of inkjet printing to precisely position materials at pre-determined locations on a substrate has opened up the possibility of high-throughput experimentation. This ability has been particularly useful in proteomics for the preparation of analysis samples and in peptide synthesis. The preparation of samples for X-ray characterization using inkjet printing has
28
also this ability. When these advantages are considered, it could be reasonable to consider the application of inkjet-fabricated biosensors being extended to the analysis of more clinically relevant micro-organisms, biomarkers and proteins. However, other ‘maskless’ techniques are gaining attention, most notably electrohydrodynamic jet printing (EHJ).[117-119] This recent technique can produce features as small as 1 μm wide lines, which is an order of magnitude smaller than inkjet printing. Naturally, the droplets produced by this technique are small also, being in the femtoliter region. Inkjet printing is responding by producing smaller droplets also, with 1 picoliter droplets being easily produced, and research being directed to the production of femtoliter droplets. Small droplet sizes are of interest since this means that even less material can be dispensed, which couples with the ongoing miniaturization seen in many applications. Recently, EHJ has been used to deposit oligonucleotides,[118] and even bovine serum albumin has been electrohydrodynamically ejected as a spray, though not yet printed.[120] One of the questions that need to be addressed is whether the droplet ejection
method of EHJ affects the material contained within the ink. Whereas inkjet printer eject their droplets from within the nozzle, EHJ printers eject their droplets from outside the nozzle. The ink in an EHJ printer forms a droplet that is attached to the nozzle. This dome of ink is charged by a wire contained within the nozzle using voltages up to 200 V,[121] which is necessary to overcome the surface tension and causes a Taylor cone to form. The droplets are ejected from the tip of the cone. Clearly, the sizes obtained by EHJ are a cause for some excitement, but the technique, which has to address problems such as susceptibility to electrical breakdown and droplet deflection during application of ink to substrates, is still in its infancy and it has not yet been applied to the full range of applications that inkjet printing has. It could be that the droplet ejection method of EHJ causes proteins to denature (of course, the opposite could also be found). While the physics of drop-on-demand inkjet fluidics has been the subject of research for decades, the same can not be said for EHJ, and much more theoretical and empirical research remains to be done in this regard. Since this technique is based on electrostatic atomization, the target must be conductive, which is also a limitation. The cost of the technique may also be a factor to consider.
Similarly, there are other non-drop-on-demand direct-write microfabrication technqiues which are gaining attention for protein dispensing, including matrix-assisted pulsed laser evaporation,[122] contact printing,[62, 123-124] dip pen nanolithography (DPN), and electrospray
29
ionization combined with Coulomb-force-directed assembly,[125] to name just a few, all are
areas of applied research, each with their own advantages and drawbacks. The outlook appears that drop-on-demand inkjet printing will continue to be a popular means of fabricating patterns of proteins on surfaces, but that other methods will also be used, with each technology developing its own respective niche, based on its advantages. Due to the wide-ranging applications of proteins on surfaces, as well as the large amount of work that has already been performed by the technique, inkjet printing will maintain a strong position in an increasingly diverse field of applied microfluidics.
When summing up, it can be seen that inkjet printing provides a number of advantages to researchers working with proteins. It can also be seen that other techniques have recently developed that appear to offer similar advantages. Such developments are to be welcomed since they encourage the established technique to improve to meet the challenge. However, the newer technology has to convince the existing users of the more mature technology that it is worthwhile to swap. As the aggregate field of microdispensing techniques continues to evolve, application niches will be negotiated for different technologies based on their demonstrated merits. As such, related emerging technologies will be worth watching, but it will also be worthwhile to see how inkjet printing responds.
1. 9 Outline of the thesis
The remaining chapters of this thesis deal with practical illustrations of the diversity of materials science applications that are achievable using reactive inkjet printing. Chapters 2 and 3 relate to the use of inkjet printing as a tool for creating geometrically controlled ionotropically reversible hydrogel structures, with dimensions that are particularly well-suited to life science applications. Chapter 4 deals with the challenges of printing living single cells, which may be thought of as microreactors, and how the dimensional attributes of inkjet can be used to make practical single cell culture microarrays. The rest of the chapters focus primarily on the use of reactive inkjet printing as a synthesis tool, starting with Chapter 5, and the preparation of molecular monolayers using a combination of reactive inkjet printing and click chemistry. Chapter 6 deals with reactive inkjet printing of irreversibly crosslinked networks, giving examples of different new phases of materials that
30
can be created by crosslinking polymerizations, divided roughly into two halves; the first part outlines an investigation into the reactive inkjet printing of polyurethanes, summarizing the first example of moisture sensitive reactive inkjet printing being untertaken, and the sort of three-dimensional microsctructures that could be achieved using it. In the second half of Chapter 6, an outline of the formulation of reactive inks for the preparation of ionic liquid gels (or ‘ionogels’) is described, where a library of ionogels is prepared and screened to find a locally optimized inkjet printable material for electronics applications. The thesis is finished with a summary bringing together the connecting themes of these contrasted topics.
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Inkjet printing of hydrogel porogens
Abstract:
Using reactive inkjet printing, hydrogel porogens were prepared and used to produce matrices with controlled, open-pore internal geometry. Taking advantage of inkjet’s ability to dispense uniform droplets in the picoliter/nanoliter ranges of volumes, droplets of sodium alginate solution were dispensed and rapidly crosslinked in the presence of calcium salts, resulting in a dispersion of ionotropically reversible, highly uniform hydrogel microbeads. These beads, which were readily soluble in the presence of ethylenediaminetetraacetic acid (EDTA), were used as soft porogens, and incorporate some unique properties as a result of being hydrogels. They offer rigorous control of pore size, such as with solid porogens, but with soft, deformable edges that ensure good pore-to-pore contact for continuous interconnectivity. However, unlike liquid or gaseous porogens, contact between the domains does result in coalescence.
Parts of this chapter have been published: J. T. Delaney, A. R. Liberski, J. Perelaer, U. S. Schubert, Soft Matter 2010, 6, 866-869.
