DIRECTED ASSEMBLY OF THREE-DIMENSIONAL STRUCTURES WITH MICRON-SCALE FEATURES

Abstract

Polyelectrolyte inks comprising a solvent, a cationic polyelectrolyte dissolved in the solvent, and an anionic polyelectrolyte dissolved in the solvent, are described. The concentration of at least one of the polyelectrolytes in the solvent is in a semidilute regime.

Description

RELATED APPLICATIONS

The present patent document is a divisional of U.S. patent application Ser. No. 11/560,610, which was filed on Nov. 16, 2006, which is a divisional of U.S. patent application Ser. No. 10/463,834, which was filed on Jun. 17, 2003. The preceding patent documents are hereby incorporated by reference in their entirety.

BACKGROUND

Three-dimensional structures with micron-scale features have many potential applications, for example as photonic band gap materials, tissue engineering scaffolds, biosensors, and drug delivery systems. Consequently, several assembly techniques for fabricating complex three-dimensional structures with features smaller than 100 microns have been developed, such as microfabrication, holographic lithography, two-photon polymerization and colloidal self assembly. However, all these techniques have limitations that reduce their utility.

Two-photon polymerization is capable of creating three-dimensional structures with sub-micron features, but from precursors that are not biocompatible. Many techniques have been developed to fabricate three-dimensional photonic crystals, but they rely on expensive, complicated equipment or time-consuming procedures. Colloidal self-assembly has also been utilized to make three-dimensional periodic structures, but controlling the formation of defects is difficult.

One fabrication technique relies on the deposition of viscoelastic colloidal inks, usually by a robotic apparatus. These inks flow through a deposition nozzle because the applied pressure shears the interparticle bonds, inducing a breakdown in the elastic modulus. The modulus recovers immediately after leaving the nozzle, and the ink solidifies to maintain its shape and span unsupported regions. The particles in the ink have a mean diameter of about 1 micron, meaning that it would be impossible for the ink to flow through a 1 micron diameter deposition nozzle without clogging or jamming. In practice, nanoparticle inks (mean diameter˜60 nm) also tend to jam nozzles smaller than 30 microns, limiting the applicability of viscoelastic colloidal inks to this length scale.

Polymeric solutions are used in nature to fabricate thin filaments. Spiders, for example, derive their silk fibers from a concentrated protein biopolymer solution that solidifies as it is drawn to form an extremely strong filament. The extensional flow of the solution aligns liquid crystal sheets in the polymer, and the solution gels by adding ions as it leaves the spinneret. This process was artificially recreated by the deposition of the recombinant spider silk biopolymer into a polar “deposition bath” to produce filament fibers with comparable properties.

SUMMARY

In a first aspect, the disclosure provides polyelectrolyte inks comprising a solvent, a cationic polyelectrolyte, dissolved in the solvent, and an anionic polyelectrolyte, dissolved in the solvent. The concentration of at least one of the polyelectrolytes in the solvent is in a semidilute regime.

In a second aspect, the disclosure provides a solid filament comprising a complex of a cationic polyelectrolyte and an anionic polyelectrolyte. The filament has a diameter of at most 10 microns.

In a third aspect, the disclosure provides a method of making a polyelectrolyte ink comprising mixing together ingredients that comprise a solvent, a cationic polyelectrolyte, and an anionic polyelectrolyte. The concentration of at least one of the polyelectrolytes in the solvent is in a semidilute regime.

In a fourth aspect, the disclosure provides a method for fabricating a filament, comprising flowing the polyelectrolyte ink through a nozzle, and contacting the ink with a deposition bath. The polyelectrolyte ink gels in the deposition bath.

In a fifth aspect, the disclosure provides a method of forming three-dimensional structure, comprising fabricating a plurality of filaments, each filament fabricated by the method set forth in the fourth aspect.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows viscosity and elastic modulus of polyelectrolyte mixtures as a function of the mixing ratio of ionizable groups at a constant polymer volume fraction (Φ_poly=0.4).

FIG. 2 shows the elastic modulus of the ink reacted in a water/IPA deposition bath as a function of IPA concentration in the deposition reservoir.

FIGS. 3A, 3B and 3C are electron micrographs of structures fabricated through the directed assembly of polyelectrolyte inks. (A) Four-layer microstructure with a missing rod that may be utilized as a waveguide in a photonic crystal. (B) Eight-layer structure with walls showing the ink's ability to form spanning and space-filling elements. (C) Radial structure showing the inks ability to turn sharp and broad angles.

DETAILED DESCRIPTION

The present disclosure provides a method of microstructure fabrication via deposition of inks that flow through a deposition nozzle of 10 micron or less, without clogging or jamming. When deposited in a deposition bath the inks solidify after leaving the nozzle. The resulting microstructures have features in the micron scale and are amenable to fabrication with biocompatible materials, and are relatively easy and inexpensive to make.

The present disclosure includes the three-dimensional fabrication of structures with micron-scale features by making use of an ink. An applied pressure forces the ink through a deposition nozzle that is attached to a moving x-y-z micropositioner, into a deposition bath that gels the ink in situ as the micropositioner moves to form a two-dimensional pattern on the substrate. The nozzle then incrementally rises in the z (vertical) direction for the next layer of the pattern. This process is repeated until the desired three-dimensional structure has been created. With this technique, any three-dimensional structure can be defined and fabricated.

The inks of the present disclosure are concentrated mixtures of oppositely charged polyelectrolytes, also referred to as polyelectrolyte complexes (PEC). The PEC contains two oppositely charged polyelectrolytes (e.g. poly(acrylic acid) and poly(ethylenimine)). One polyelectrolyte is preferably larger than the other, and the concentration of the larger polyelectrolyte is preferably within the semidilute regime: the concentration is above the concentration c* that separates the dilute from the semidilute concentration regime. Below c*, in the dilute regime, the mixture of polyelectrolytes forms particles rather than the single phase fluid needed for the deposition of a continuous filament. Above c*, in the semidilute regime, polymer coils strongly overlap with each other, and the mixture of electrolytes may be used for structure deposition.

The ink viscosity is preferably in the range that allows consistent, controllable flow at a modest applied pressure. Preferred viscosity values vary between at least 0.05 Pa*sec to at most 600 Pa*sec. More preferred viscosity values are at least 0.1 Pa*sec to at most 150 Pa*sec. Yet more preferred viscosity values are at least 1 Pa*sec to at most 20 Pa*sec. Moreover, the ink undergoes a rapid solidification reaction when it comes in contact with the deposition bath that allows the extruded filament to maintain its shape while spanning unsupported regions of the structure.

Examples of polyelectrolytes that may be used in PEC are poly(acrylic acid), poly(ethylenimine), poly(styrene sulfonate) poly(allylamine) hydrochloride, poly(diallyldimethyl ammonium chloride), poly(4-vinyl pyridine), and cationic or anionic surfactants. Electrically or optically active classes of polymers, for example polyacetylene, polyaniline, polypyrrole, polythiophene, poly(3,4 ethylenedioxythiophene) (PEDOT), NAFION® (Du Pont, Wilmington, Del.), polyphenylene vinylene, polyphenylbenzenamine, sulfonated poly-p-phenylene azobenzene dye and other organic dyes may be used, and are well suited to applications involving organic LEDs and circuits. The parent polymers of some of these classes of polymers do not contain charged groups, however, copolymers and derivatives of these classes do; for example charged groups may be introduced through monomers containing substituents (which may be protected until after synthesis of the polymer), or by derivatizing reactive groups (such as hydroxyl groups, or electrophilic addition on phenyl rings).

For biochemical, molecular biological and biomedical applications, such as biocatalysis, gene manipulation and tissue engineering, biological electrolytes may be used. Example biological polyelectrolytes are polynucleotides, such as DNA and RNA, peptides, proteins, peptide nucleic acids, enzymes, polysaccharides such as starch and cellulose, acidic polysaccharides such as hemicelluloses (for example arabinoglucuronoxylan), basic polysaccharides such as poly-(1,4) N-acetyl-D-glucosamine (chitosan), galactans such as agarose, polyuronides such as alginic acid, carrageenans, hyaluronic acid, collagen, fibrin, proteoglycans, polylactic acid, polyglycolic acid, copolymers of organic acids, cationic lipids. Biological polyelectrolytes with both positive and negative charges, for instance zwitterions such as polycarboxybetaine, may also be included in the ink compositions.

Bioactive molecules may also be incorporated in the ink, for example charged or neutral nutrient molecules, molecular messengers such as growth stimulants, and cellular adhesion molecules. Molecular probes for biomolecules such as cellular lipids or cellular membrane proteins, cellular components such as ion channels and receptors, or organelles such as mitochondria or lysosomes may also be added.

Smaller organic and inorganic species can also be incorporated into the inks, to amounts that do not deleteriously affect the rheological properties of the ink. Examples include nanoparticles, quantum dots, charge neutral polymers, organometallic precursors and biomolecules. These species may interact with the polyelectrolytes to aid in the gelation or remain inert in the ink, depending on their ionic nature. Also, many other polymers may be made into polyelectrolytes through functionalizing the polymer backbone with charged moieties, for example amino groups, sulfonate groups, and carboxylic groups.

The molecular weight of the larger polyelectrolyte is preferably high enough to facilitate chain overlap (preferably at least 5000 daltons) but also low enough to form a concentrated ink with a viscosity that enables flow at moderate pressures (preferably at most 100,000 daltons). The concentration of the ink is preferably high to avoid deformation of the structures upon drying. A typical polymer concentration ranges from at least 5% to at most 95% by weight. More preferably, the concentration varies from at least 25% to at most 75% by weight. Yet more preferably, the concentration varies from at least 35% to at most 45% by weight. Most preferable concentrations range from at least 38% to at most 42% by weight.

The larger polyelectrolyte and the smaller polyelectrolyte are preferably mixed together in a ratio such that one of the charge groups is in excess (usually the charge group of the larger polymer), yielding a mixture away from a stoichiometric (1:1) cationic:anionic group ratio. In the vicinity of this ratio, the strong interactions between complementary polyelectrolytes may lead to the formation of kinetically stable, inhomogeneous aggregates, and the complex may form two phases, a polymer-rich aggregate and a polymer-poor fluid.

Once the polyelectrolytes and solvent have been chosen, a phase diagram can be developed relating the ratio of cationic to anionic groups as a function of overall polyelectrolyte concentration (in the chosen solvent), with the goal of determining the range for homogeneous inks. This range will be above the dilute/semidilute transition of the larger polymer and away from a stoichiometric (1:1) cationic:anionic group ratio. The viscosity of the homogeneous inks increases as the polymer concentration increases and as the mixing ratio approaches 1:1. The viscosity may thus be controlled for the deposition of inks through a variety of nozzle sizes.

A deposition bath is selected to fabricate three-dimensional structures through a rapid solidification reaction. In these polyelectrolyte inks, the reaction will occur by increasing the strength of the attractions between the oppositely charged polyelectrolytes. This can be achieved, for example, through pH changes, ionic strength changes, solvent composition changes, or combinations of more than one change. The reaction produces a filament that is strong enough to maintain its shape while spanning unsupported regions in the structure, but also soft enough to allow the filament to adhere to the substrate and flow through the nozzle consistently.

Deposition baths that induce gelation through pH changes are generally used when the polyelectrolytes contain acidic and/or basic charged groups. The pH change eliminates the excess of one of the charge groups, for instance by ionizing acidic groups that are neutral at the pH of the ink. This yields a mixture with a stoichiometric (1:1) cationic:anionic group ratio that gels into a filament.

In addition, the pH of the deposition bath may be selected in order to induce partial dissolution of the deposited filament, while the shape is maintained. The pH of the bath lowers the bond strength between the oppositely charged polyelectrolytes, leading to the dissolution. The structures have a residual charge on the surface, and may be used for the adsorption of charged nanoparticles.

Coagulation may also be achieved through changes in solvent composition. For example, an aqueous ink may be deposited in a deposition bath containing a relatively apolar solvent such as an alcohol. The resulting drop in dielectric constant leads to an increase in the coulombic attractions between the polyelectrolytes. Also, an apolar solvent that is a poor solvent for the polyelectrolytes may be chosen, leading to increased polyelectrolyte/polyelectrolyte bonding. The reaction yields a polyelectrolyte complex precipitate with a positive:negative charge ratio closer to 1:1 than in the unreacted ink, but not to the extent of the pH induced reaction. The structures have a residual charge on the surface, and may be used for the adsorption of oppositely charged nanoparticles.

Moreover, the mechanical properties of the deposited ink are dependent on the composition of the deposition bath. As illustrated in FIG. 2, different percentages of apolar solvents generally yield filaments of varying stiffness.

An apparatus for depositing the ink may be manufactured by connecting a deposition nozzle with a diameter of preferably at least 0.1 microns to at most 10 microns to a micropositioner, for example a computer controlled piezoelectric micropositioner, and an ink reservoir. These micropositioners are used in a variety of devices, such as scanning tunneling microscopes, and are commercially available. Pressure pushes the ink through the nozzle, or two or more nozzles, and the micropositioner controls the deposition pattern of the filament. Alternatively, the nozzle (or nozzles) may be static, while the stage holding the substrate on which the microstructure is formed may be controlled by the micropositioner. In another configuration, both the nozzle and the stage may each be controlled by its own micropositioner. Multiple substrates are also possible when multiple nozzles are present. The assembly of structures is then preferably performed using patterns created in a computer aided design computer program coupled to the micropositioner.

There are many applications for solid PEC structures fabricated with these materials and methods. The structure may be infiltrated with a high refractive index material and subsequently the PEC structure redissolved to form photonic crystals. The ability of the ink to span distances renders it possible to engineer defects (for example cavities or waveguides) into the structure for functional photonic band gap materials. The highly porous structures could be used for membranes that selectively allow small molecules to flow through at a faster rate. Also, screens may be prepared that do not allow cells, or cells larger than a certain size, to flow through. Screens of this type may be used, for instance, to separate smaller cells from larger cells in a blood sample. They may also be used for drug delivery systems where a range of porosities is necessary for controlled release.

The charged complexes may be used as tissue engineering scaffolds for cell adhesion and growth. For instance, copolymers of poly(L-lactic acid) and poly(L-glycolic acid), both anionic polyelectrolytes that have been FDA approved as biodegradable polymers, may be combined with one or more cationic polymers, such as chitosan, to form a biocompatible ink for tissue engineering applications. To promote cell growth throughout the structure, proteins and sugars may be added to the ink to be released as the polyelectrolytes dissolve.

Biologically interesting molecules may also be attached to the microstructures. Examples of these molecules include nucleic acids, polypeptides and other organic molecules. Nucleic acids include polynucleotides (having at least two nucleic acids), deoxyribonucleic acid (DNA), such as expressed sequence tags (ESTs), gene fragments or complementary DNA (cDNA), or intron sequences that may affect gene transcription, such as promoters, enhancers or structural elements. However, the nucleic acid need not be related to a gene or gene expression, as aptamers (small nucleic acid sequences that specifically bind to a target molecule) can also be used. Ribonucleic acids (RNA) may also be used, such as messenger RNA (mRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA). Nucleic acids may also be modified; for example, such as substituting a nucleic acid with a non-naturally occurring one, such as inosine. Chemical modifications of nucleic acids, such as those that may confer stability or facilitated immobilization upon a substrate, may also be used. Another example of a modified nucleic acid is a peptide nucleic acid (PNA), which is a nucleic acid mimic (e.g., DNA mimic) in that the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs allows for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols. Nucleic acids attached to the microstructures may be used, for example, in diagnostic and prognostic assays, gene expression arrays, pharmacogenomic assays, etc.

Polynucleotides may be linked to the microstructures through thiol-mediated self-assembly attachment to gold nanoparticles incorporated into the microstructures. The gold may be incorporated into the microstructures either by addition to the undeposited ink mixture or by attachment to the microstructures after deposition, for example via sulfhydril groups present in the ink.

Polypeptides, having at least two amino acid residues, may find use on or within the substrates of the application. Examples of classes of polypeptides include antibodies and derivatives, protein hormones (e.g., human growth hormone and insulin), extracellular matrix molecules, such as laminin, collagen or entactin; polypeptides involved in signaling, such as phosphatases and kinases; receptors, such as dopamine receptors and hormone receptors (advantageously may be attached in the native format, or in the case of homodimers, trimers, etc., mixed with other polypeptide chains or as single chains), etc. Attaching polypeptides to the substrates of the disclosure allow for a wide variety of applications, including drug screening, diagnostic and prognostic assays, assays that resemble enzyme-linked immunosorbent assays (ELISAs), proteomic assays and even cellular adhesion studies.

Organic molecules also find use on the microstructures. For example, steroid hormones, such as estrogen and testosterone may be attached. Such couplings facilitate for screens for molecules that bind these molecules, such as antibodies or aptamers. Likewise, candidate small molecule antagonists or agonists may be attached to facilitate pharmaceutical screening.

Entities such as prions, viruses, bacteria, and eukaryotic cells may also be attached. Prions are misfolded protein aggregates that can propagate their misfolded state onto native proteins; examples include those aggregates that cause mad cow disease (bovine spongiform encephalopathy (BSE)) or Creutzfeldt-Jacob disease. Examples of viruses include herpes simplex, orthopoxviruses (smallpox) or human immunodeficiency virus. Bacteria that are of interest may include Vibrio cholera, Clostridium perfringens, or Bacillus anthracis (anthrax). Eukaryotic cells, such as those isolated as primary cultures from subjects or plants, or from cell lines (e.g., those available from the American Type Culture Collection (ATCC); Manassus, Va.), may be immobilized onto the microstructures for a variety of purposes, including screens for pharmaceuticals, investigations into cell-substrate adhesion, or for the binding of various molecules.

Any ink that gels through a solvent change may be used to assemble three-dimensional structures from electrically, optically or biologically active polymers. Inorganic structures may also be fabricated by using sol-gel precursors to produce, for example, sensors or template-free photonic band gap materials.

EXAMPLES

1) Ink Mixtures

A linear polyacid, poly(acrylic acid) (MW˜10,000) and a highly branched polybase, poly(ethylenimine) were combined in an aqueous solvent, yielding solutions with a polymer fraction Φ_poly=0.4. When these polyions were combined, the carboxylate groups of poly(acrylic acid) (PAA) form ionic bonds to the amine groups of poly(ethylenimine) (PEI). The polymers were initially mixed under mildly acidic conditions (pH˜3.6), where the partial charge on the PAA let only a fraction of the potentially ionizable groups participate in complexation. The Φ_poly was maintained constant, and different PAA to PEI ratios yielded mixtures with varying rheological properties, as illustrated in the phase diagram of FIG. 1. In this figure, the values on the left and the right y-axes indicate values for pure PAA and pure PEI at Φ_poly=0.4 The dilute-semidilute crossover concentration c* for PAA is indicated on the bottom x-axis. The two-phase region consists of a dense, polymer-rich phase, and a fluid-like, polymer-poor phase, and data could not be obtained in this regime. As the ratio approaches the two-phase region, the elastic modulus and the viscosity of the mixtures increases.

A homogeneous, single phase was observed at mixing ratios in the PAA and PEI rich regions. The charge imbalance forms a non-stoichiometric, hydrophilic complex. The two-phase region, near stoichiometric mixing ratios, comprises a dense, polymer-rich phase with a stoichiometric, hydrophobic complex and a fluid-like, polymer-poor phase.

The viscosity differences observed at different mixing ratios may be utilized to assemble structures at different length scales. At small nozzle sizes, a lower viscosity ink may be deposited at modest applied pressures, whereas larger nozzle sizes generally require more viscous inks in order to obtain flows with controlled rates.

2) Ink Deposition Apparatus

Inks prepared as described in example 1 were loaded in a deposition apparatus for microstructure fabrication. The apparatus comprised a NanoCube™ XYZ NanoPositioning System (Polytec PI, Auburn, Mass.) controlling μ-Tip (World Precision Instruments, Sarasota, Fla.) deposition nozzles, and the ink was dispensed from the apparatus by a Model 800 ULTRA Dispensing System with a 3 ml ULTRA Barrel Reservoirs (EFD, Providence, R.I.).

3) Fabrication of Structures in an Isopropanol and Water

An ink prepared according to the procedure of example 1, with a Φ_poly=0.4, a PAA:PEI ratio of 5.7:1, was deposited, at a velocity of 20 microns/second and through a 1 micron nozzle, in a deposition bath containing a mixture of isopropanol (IPA) and water. The gelation occurs due to a decrease in solvent quality for the polyelectrolytes and an increase in the coulombic attractions between the ionizable groups, yielding a reacted ink filament. NMR spectroscopic data (not shown) showed no discernable difference in the types of bonds in the reacted and unreacted complexes, indicating that the reaction only causes a change in the number and strength of the bonds. The mechanical properties of the reacted ink were highly dependent on the deposition bath, as illustrated in FIG. 2.

4) Fabrication of Structures in a Water Deposition Bath

The experiment of Example 3 was repeated, this time using an ink with a PAA:PEI ratio of ˜4.8 and a deposition bath of deionized water. The pH change eliminated the excess of the groups bearing a positive charge by ionizing acidic groups that were neutral at the pH of the ink. This yielded a mixture with a nearly stoichiometric cationic:anionic group ratio that gelled into a filament (Table 1).

TABLE 1
				(−:+) charge
Polyelectrolyte	Carbon	Hydrogen	Nitrogen	ratio
PAA	39.48	4.80		NA
PEI	52.04	11.78	31.68	NA
Unreacted PEC	40.12	5.21	2.27	4.8:1
Reacted PEC	41.92	5.58	2.90	1.1:1

4) Microstructures

FIGS. 3A to 3C show structures fabricated with a 1 micrometer nozzle in an 83% IPA (balance water) deposition bath. FIG. 3A shows an FCT structure with a missing filament in the middle that could be used as a waveguide in a photonic crystal. FIG. 3B shows an 8-layer structure with walls, showing the ability to form solid structures as well as spanning elements. FIG. 3C shows a radial structure with porosity at multiple length scales. The reacted complex is capable of spanning lengths much greater than the filament diameter. Waveguides and radial structures were fabricated, showing the ability to fabricate structural features with tight or broad angles. Periodic structures with different feature sizes were also fabricated, wherein the feature size of a structure is the diameter of its thinnest filament, obtained with different nozzles and inks (Φ_poly=0.4 and PAA:PEI ratio˜5.7:1; Φ_poly=0.4 and PAA:PEI ratio˜5:1; Φ_poly=0.43 and PAA:PEI ratio˜2:1).

If the deposition occurs in a slightly acidic reservoir, partial dissolution of the complex occurs, while the shape is maintained, leading to highly porous structures with lower elasticity. The structures have a residual negative charge on the surface, and may be used for the adsorption of nanoparticles.

The microstructures may also undergo thermal treatment and maintain their integrity. For example, the microstructures were heated at 5° C./min to 240° C. in air. The temperature was held at 240° C. for 30 minutes, and then cooled at 5° C./min. The structures maintained their original shape and became harder than prior to the heating. This hardening may be due also to heat-induced inter-polyelectrolyte bond formation, for example amide bonds formed between the carboxyl groups of the PAA and the amine groups in the PEI.

Claims

1. An apparatus for the fabrication of microstructures, the apparatus comprising:

(1) a nozzle comprising an opening having a diameter of at most 10 microns;

(2) a stage below the nozzle for supporting a substrate;

(3) a micropositioner operably connected to at least one of the nozzle and the stage; and

(4) an ink reservoir in fluid communication with the nozzle.

2. The apparatus of claim 1, further comprising a pressure dispensing system operably connected to the ink reservoir.

3. The apparatus of claim 1, wherein the micropositioner is a computer-controlled micropositioner.

4. The apparatus of claim 1, wherein the opening of the nozzle has a diameter of at most 1 micron.

5. The apparatus of claim 1, wherein the opening of the nozzle has a diameter of at least 0.1 micron.

6. The apparatus of claim 1, further comprising a plurality of nozzles.

7. The apparatus of claim 6, further comprising a plurality of substrates on the stage.

8. The apparatus of claim 1, further comprising a substrate on the stage, the substrate comprising a deposition bath.

9. The apparatus of claim 8, wherein the deposition bath is configured to induce gelation of an ink flowed through the nozzle.

10. The apparatus of claim 8, wherein the deposition bath comprises an aqueous solvent.

11. The apparatus of claim 8, wherein the deposition bath comprises an apolar solvent.

12. The apparatus of claim 11, wherein the apolar solvent comprises an alcohol.

13. The apparatus of claim 12, wherein the deposition bath comprises a mixture of isopropyl alcohol and water.