WO2013148938A1 - Improvements to microplates and methods for protein crystallization and biotechnology - Google Patents

Abstract

Devices and methods for manual and high-throughput protein crystal growth and growth of other biological and organic crystals. A microplate includes a plurality of cells and a frame that defines the cells in the microplate. In each cell there is at least one well open at top. Each well in a cell may be enclosed at bottom, or it may be open at bottom, in which case the well bottom may be sealed by a separate part, which may be, e.g., a separate film or plate (e.g., of plastic, glass or metal) or a molded part.

Description

IMPROVEMENTS TO MICROPLATES AND METHODS FOR PROTEIN CRYSTALLIZATION AND BIOTECHNOLOGY REFERENCE TO RELATED APPLICATIONS The instant application claims priority to US provisional application S/N 61/617,102 filed on 03/29/2012, the subject matter of which is incorporated by reference herein. The invention pertains to the field of biotechnology. More particularly, the invention pertains to improvements to microplates, including those used for protein crystal growth. Many fields of bioscience and biotechnology use microplates for screening and other experiments. Microplates generally consist of a large number (e.g., 24, 96, 386, 1536) of identical cells arranged in a regular (usually rectangular) array. Each cell contains one or more wells or reservoirs into which liquids or other samples of interest are dispensed. The microplates are generally made by injection molding plastic. After dispensing samples into the wells, the top of the microplate is often sealed to protect the experiment from the environment. Most microplate designs conform to ANSI standards established by the Society for Biomolecular Sciences (SBS). A specific application of microplates is in the fields of structural biology and X-ray crystallography, where they are used to grow crystals of proteins, nucleic acids, viruses, and other biomacromolecular complexes, and to explore the solubility of proteins in different solutions. Obtaining crystals of suitable size and quality for X-ray diffraction studies remains an important bottleneck in determining structures of biological macromolecules. Solution conditions (pH, salt type and concentration, protein concentration, concentrations of cryoprotectants and other additives) that yield crystal growth must be identified, and then optimized to yield crystals with adequate diffraction resolution for structure determination. Figure 1 shows a typical protein crystallization microplate, and Figure 1(c) shows a typical cell in such a microplate. Each cell in the microplate generally has one or more small wells for protein solution and a larger well for the "reservoir" solution. Vapor diffusion is the most common method for growing crystals of proteins, viruses and biomolecular assemblies, as well as of small molecule compounds that may be useful as drugs. In vapor diffusion growth, each reservoir well is filled with a protein-free solution, and a drop of protein solution is deposited on the bottom of one of the smaller wells. The microplate (and thus each cell) is then sealed using a plastic film. Typical volumes of reservoir solution are 20-200 microliters, and typical volumes of protein solution are 0.2-2 microliters. The air spaces above the reservoir and protein wells, within any given cell, are open to each other, allowing vapor to flow between them. The reservoir solution initially has a lower water vapor pressure than the protein drop. Water evaporates from the protein drop and condenses in the reservoir until the vapor pressures reach equilibrium. Water evaporation from the protein drop gradually increases the protein concentration in the protein drop. In favorable circumstances, this leads to crystal nucleation and growth. Some manufacturers of plates for protein crystallization include Greiner Bio-One International (Austria), Corning (Corning, NY), Art Robbins Instruments (Sunnyvale, CA), Hampton Research (Aliso Viejo, CA), Neuroprobe (Gaithersburg, MD) and TTP LabTech (UK). Together, more than 200 different designs for crystallization plates are available. Other approaches to high-throughput protein crystallization are being pursued. For example, Fluidigm (San Francisco, CA) and Emerald Biosystems (Bainbridge, WA) have commercialized platforms based on microfluidic chips. Although these allow crystallization with very small volumes, the chips are expensive compared with conventional microplates, they require specialized and very expensive hardware for loading, and the chip-hardware combination provides less flexibility in design of crystallization experiments. They are also incompatible with the SBS standards. An important direction in recent years has been the development of methods for examining crystals using X-rays without removing the crystals from the plate or device in which they are grown. Drops have been dispensed into nylon loops, onto thin films, or into X- ray transparent glass capillaries, and the crystals that have grown have been examined by directing X-rays through the film, loop or capillary without removing the crystals. Oxford Diffraction (since acquired by Agilent) developed a special X-ray machine with a vertical X- ray beam for examining crystals in conventional SBS microplates. Greiner Bio-One in collaboration with NatXray (France) has developed microplates with thin (250 micrometer) windows to reduce background scattering in such applications. Problems with Current Microplate Technology Microplates are held in a horizontal orientation during liquid dispensing and routine use. If current protein crystallization microplates are rotated toward the vertical or if they are inverted, within each cell the contents of the wells will spill out and mix. Similarly, mixing can occur if the plates experience sharp accelerations. Mixing corrupts experiments. In the case of protein crystallization, early mixing of reservoir and protein solutions can lead to abrupt precipitation or to nucleation and growth of very large numbers of unsuitably small crystals. Consequently, most current microplates must be kept near the horizontal and handled very gently. This handling restriction limits the usefulness of current plates, for several reasons. First, since current microplates cannot be inverted, crystallization can only be performed in the "sitting drop" configuration. Under the influence of gravity, crystals that nucleate within the protein drop will sediment onto the supporting surface. Often they adhere to this surface, making retrieval difficult. If the plates could be inverted to the "hanging drop" configuration, crystals would sediment to the drop-air interface, from which they could be easily harvested for subsequent study. 75 Second, since plates cannot easily be rotated to the vertical without the danger of mixing

76 well contents, inspection of each cell using X-rays and other electromagnetic probes is much

77 more difficult. Nearly all X-ray sources used in crystallography - including tube and rotating

78 anode lab sources and synchrotron sources - produce horizontal X-ray beams, so that plates

79 must be rotated to the vertical for X-ray inspection of the contents of each cell. Infrared and

80 UV spectrometry are also typically performed using horizontal illumination. In protein

81 crystallization, the protein drops often contain precipitate and salt crystals and are often

82 covered by "skins" formed from polyethylene glycols or denatured protein. Visually

83 identifying crystalline protein, especially when the crystals are only microns in size, can be

84 extremely difficult. In situ inspection using X-rays provides the most reliable crystal detection

85 and assessment of crystal quality.

86 Third, with sufficiently X-ray transparent plate materials, in favorable cases X-ray

87 crystallography and structure determination can be performed in situ, on a crystal residing in

88 the plate. However, this in general requires that the crystal and thus the plate be rotated by

89 typically 60 or 90 degrees about axes perpendicular to the X-ray beam. This is feasible with

90 current plates only for crystals with certain orientations.

91 Fourth, in almost any non-automated handling of microplates, accidental jolts and other

92 large accelerations due to mishandling are common. The resulting corruption of the

93 experiment often necessitates that it be repeated.

94 Finally, once they are filled with solutions, current plates cannot be easily transported,

95 e.g., between nearby buildings, or in a car or airplane from a home laboratory to another

96 laboratory or to a synchrotron X-ray source. Current plates cannot be shipped by mail or

97 private courier from one location to another. Plate tilting and impulsive accelerations during

98 shipping and handling cause liquids contained in each well to spill out. This can disperse the

99 liquid, increasing its total surface area, which can have a large impact on the rate of vapor

100 diffusion between liquids in each well, on the rates of crystal nucleation and growth, and on

101 the protein/biomolecule oxidation, degradation and/or crystal nucleation that occur at air- 102 liquid interfaces. Plate tilting and impulsive accelerations can also cause mixing of solutions

103 in wells contained within a given cell, corrupting the experiment. In protein crystallography,

104 a growing fraction of X-ray data collection is performed remotely. Crystals mounted in

105 special holders are frozen to near T=77 K and shipped to synchrotron sources for

106 measurements. Shipping of crystallization microplates to synchrotrons for remote data

107 collection is not currently feasible.

108 In addition to the above problems, current microplates for crystallization suffer from an

109 additional problem. When liquid is dispensed into the reservoir well, it typically does not fill

110 the well uniformly, especially if the well is much longer in one direction than another. The

111 detailed behavior depends on how the aqueous solution wets the plastic or other material used

112 to form the well, i.e., on the contact angle and contact angle hysteresis. If the contact angle is

113 large, the liquid may "ball up", residing in the center of the well and avoiding its corners. It

114 may be drawn to and wet a corner the well. If the contact angle is small (as for solutions

115 containing alcohols) it may climb up the sides of the well. In all cases, this tends to reduce the

116 total volume of liquid that can be easily dispensed into a well of given dimensions without

117 overflow, and without the fluid contacting the top sealing film during the sealing of the plate.

118 It also results in irreproducible liquid-air interface areas for the same liquid volume. Since the

119 exposed surface area affects the rate of evaporation from the surface, this may affect the rate

120 of vapor diffusion between wells and thus contribute to irreproducibility in crystallization.

121 Incomplete filling of reservoir wells also necessitates the use of wells that are much

122 deeper or taller than necessary. Since the minimum thickness of a microplate is determined by

123 the reservoir well height / depth, this in turn limits the minimum plate thickness. Minimizing

124 plate thickness is desirable to minimize storage requirements for microplates. It is also

125 desirable to facilitate X-ray inspection on commercial X-ray crystallography apparatus and at

126 synchrotron X-ray sources, since the available space for a plate in these set-ups is typically

127 quite constrained. 128 None of the currently available crystallization microplates include features that address

129 these problems.

130 The present invention teaches devices and methods for manual and high-throughput

131 protein crystal growth and growth of other biological and organic crystals, as well as for other

132 applications in biotechnology. In one embodiment, a microplate comprises a plurality of cells,

133 and a frame that defines the cells in the microplate. In each cell there is at least one well open

134 at top. Each well in a cell may be enclosed at bottom, or it may be open at bottom, in which

135 case the well bottom may be sealed by a separate part, which may be, e.g., a separate film or

136 plate (e.g., of plastic, glass or metal) or a molded part. Current microplates for protein

137 crystallization typically contain between 2 and 4 wells per cell.

138 The present invention provides means for allowing vapor communication between

139 wells in the same cell while inhibiting liquid transfer between the wells. This means for vapor

140 communication also allows some control over the rate of vapor transfer and equilibration

141 between wells.

142 The present invention provides means for encouraging liquid drop spreading and

143 complete and more uniform filling of a well to a given height, for liquids with a variety of

144 surface tensions and contact angles.

145 The present invention thus provides means for maximizing liquid volume in a given

146 well area, and thus for minimizing the well height required to hold a given volume in a given

147 area. This allows the plate height to be minimized. Storage requirements for plates can then

148 be reduced.

149 In microplates where one or more well bottoms or bottom sealing films are X-ray

150 transparent, the smaller plate height allowed by the present invention allows incident and

151 transmitted X-ray diffraction angles over a larger angular range without X-rays intercepting

152 microplate materials. 153 The smaller well height also maximizes the range of harvest angles from which

154 crystals or other samples can be retrieved from drops placed in a well whose bottom coincides

155 with the bottom surface of the plate.

156 The present invention also prevents liquid from climbing up the side wall of a well to

157 the top of the well wall. This makes sealing the top surface of the plate more reliable and

158 secure.

159 The present invention provides means for inhibiting liquid motion and liquid contact

160 of a top sealing surface when the plate is tilted, inverted or accelerated. This in turn allows the

161 plate to be inverted for hanging drop crystallization, to be more roughly handled, and to be

162 transported without mixing of solutions within the plate.

163 The present invention allows these features to be achieved while providing easy filling

164 of all wells using standard liquid handlers and pipetters, and with dispensing patterns similar

165 those used to fill existing commercial plates.

166 The wells within each cell are connected by communication channels on the top

167 surface of the microplate. The cross-sectional areas of these channels are sufficiently small

168 that, when at least one of the wells is filled with fluid, the hydrostatic fluid pressure created

169 when the microplate is rotated to any orientation is insufficient to drive fluid flow through the

170 channels from one well to the other. The communication channels are also sufficiently small

171 that liquid splashing and fluid pressures generated within a well during routine handling or

172 typical mishandling do not drive appreciable fluid flow through the channels. The channels

173 may be straight. They may also be curved. They have they may have jogs or offsets that

174 prevent ballistic liquid motion through them. The invention is further comprised of a liquid

175 retention ledge or ridge or aperture that extends around the interior perimeter of one or more

176 of the wells, and projects outward from the wall of the well toward the well center. The open

177 area of the aperture has a diameter that is smaller than the well diameter but is of sufficient 178 length and width to allow standard diameter/profile pipette tips and other liquid dispensing

179 tips to be inserted into the well and to contact the bottom of the well.

180 During liquid dispensing and well filling, liquid wetting to the lower surface of the

181 ledge and contact line pinning by the interior perimeter of the aperture formed by the ledge

182 facilitates spreading of the liquid across the bottom of the well and uniform filling of the well.

183 Without the ledge, a liquid with a contact angle near 90 degrees (typical of alcohol-

184 free aqueous solutions on plastics) will tend to form a hemispherical drop on the bottom of the

185 well. This drop will fill only a fraction of the well volume - especially for wells that are

186 rectangular or elliptical - and will project upward close to the top surface of the well, where it

187 may contact the film used to seal it. Any such contact can prevent proper sealing of the film to

188 the top surface of the plate, and must be eliminated.

189 With the ledge, as liquid is dispensed the hemispherical liquid drop grows in height

190 until it contacts the bottom surface of the ledge. Liquid then spreads laterally beneath the

191 ledge, more uniformly filling the volume below the ledge, before eventually emerging

192 through the aperture when the well is overfilled. For high surface tension liquids like water

193 and salt-containing aqueous buffer solutions, the total liquid volume that can be dispensed in a

194 well of a given height and base area can then be maximized.

195 The height of the wells is an important parameter in microplate design. The well

196 height limits the minimum plate height, and thus determines plate storage volume

197 requirements. Small well heights make it easier to dispense liquid into the bottom of a well

198 and to retrieve, e.g., crystals that may grow in drops dispensed on the bottom of a well. They

199 also increase the range of possible incident and diffracted X-ray beam angles that do not

200 intercept plate materials during in situ X-ray inspection of well contents. Ledges/apertures as

201 described here allow the liquid volume that can be dispensed in a given well volume to be

202 increased, and thus allow the well height for a given liquid volume to be reduced. 203 When the well is filled until the liquid touches the bottom surface of the ledge, liquid

204 then spreads across the aperture. The surface area of the liquid in the well that is exposed to

205 the air above is thus defined by the aperture, rather than by the larger and otherwise

206 irreproducible drop shapes typically formed by dispensed liquids. This may lead to more

207 reproducible equilibration between wells and more reproducible crystallization outcomes.

208 Wetting and contact line pinning at the aperture's inner surface, and near complete

209 filling of the volume below the ledge with liquid strongly inhibit liquid flow through the

210 aperture and out of the well when the microplate is tilted or accelerated. Liquid flow out the

211 aperture requires that air enter through it, which is strongly inhibited by the small aperture

212 dimensions and the liquid surface tension. Positioning the top surface of the ledge/aperture a

213 finite distance below the microplate's top surface prevents any liquid that bulges through the

214 aperture during tilting, inverting and acceleration from contacting the top sealing film and

215 spreading. Together, the combination of the retaining ledge/aperture within a well and

216 communication channels connecting the wells strongly inhibit liquid transfer between wells.

217 This allows the microplates to be rotated to any orientation without liquid transfer, to survive

218 routine handling and mishandling, and to be transported and shipped without liquid transfer.

219 The retaining ledge/aperture and the communication channels thus allow microplates

220 to be produced that have additional functionality and allow new methods for using

221 microplates.

222 Fig. 1 (a) and (b) show top and side views of a 96 cell microplate used in protein

223 crystallization, manufactured by Swissci. Fig. 1 (c) shows a schematic of one cell in such a

224 microplate with a small protein drop well at left and a much larger reservoir solution well at

225 right.

226 Fig. 2 (a) shows a top view of a microplate in one embodiment of the present

227 invention. Fig. 2 (b) and (c) show top and cross-sectional side views of a single cell of a 228 microplate in this embodiment, with communication channels 17, liquid retention aperture 15

229 and liquid retention ledge 16.

230 Fig. 3 shows example alternative embodiments of the communication channels that

231 connect wells within a single cell according to the present invention.

232 Fig. 4 (a) and (c) shows side schematic views of two alternative embodiments of a

233 well with a liquid retention ledge and aperture according to the present invention. Fig. 4 (b)

234 and (d) show fully rendered views of cells in a microplate, with the right-hand well in each

235 cell having liquid retention ledges; the bottom of the wells are sealed using a separate part

236 which may be a film, a plate, or a separately molded part. Fig. 4 (e) shows a microplate

237 similar to 4(b) but with an integral sealed bottom for each well. Figs. 4(f) and 4(g) show

238 variants of 4(e) in which the left-hand well depth is smaller than the right-hand well depth.

239 Fig. 5 (a) schematically shows how water spreads during filling of a well in a

240 conventional microplate. Fig. 5(b) shows how water spreads during filling of a well with a

241 liquid retention ledge and aperture, according to one embodiment of the present invention.

242 Fig. 6 (a) shows photographic images of water spreading during well filling of a

243 standard commercial microplate. Fig. 6 (b) shows photographic images of water spreading

244 during filling of a microplate with a liquid retention ledge and aperture, in one embodiment of

245 the present invention

246 Fig. 7 shows one liquid-containing well in one embodiment of the present invention,

247 when it is inverted.

248 Fig. 8 shows alternative embodiments of the liquid retention ledge and aperture in a

249 well of a microplate according to the present invention.

250 The present invention consists of modifications to microplates such as those used in

251 protein crystallization and screening that strongly inhibit the transfer of liquid between wells 252 within each cell of the microplate while allowing vapor communication between the wells.

253 This allows the microplates to be used in any orientation and to be handled, transported and

254 shipped without mixing of liquids in the connected wells. This also allows the reservoir well

255 volume and height to be minimized, and also increases the consistency of reservoir fluid

256 surface area.

257 Vapor communication channels.

258 As illustrated in Fig. 1, in current crystallization microplates, within each cell the

259 wells for reservoir solution 5 and for protein drops 10 are open to each other, allowing free

260 exchange of vapor between the reservoir well and the protein well(s). This is accomplished by

261 leaving a gap 12 between the top of the barrier or wall between wells and the top surface 7 of

262 the microplate, as shown in Fig. 1(c), the top surface being sealed after microplate loading

263 using a plastic film. Typical dimensions of this gap are approximately 1 mm high by 8 mm

264 wide. This large gap allows liquid to easily flow between wells when the microplate is tilted,

265 inverted or bumped.

266 Various approaches could reduce liquid transfer while allowing vapor communication

267 between wells. For example, a barrier of micro porous material (e.g., filter material) could be

268 used to fill the gap between the top of the barrier wall and the top surface of the microplate.

269 Our preferred embodiments, schematically illustrated in Fig. 2, involve extending the top of

270 the barrier wall between protein drop well 20 and reservoir well 15 within a given cell to the

271 top of the microplate, and forming small-cross-sectional area communication channels 17 in

272 the top surface of the plate, which provides the sealing surface of the cell. Fig. 3 illustrates

273 alternative embodiments of the channels. In Fig. 3(a), there is a single communication channel

274 25 disposed at one end of the reservoir well, so that any liquid transfer that does occur

275 between wells occurs away from the drops which may be disposed near the center of the

276 adjacent well. There may be more than one communication channel (Fig. 3(b)), and the

277 channels can be angled (Fig. 3(c)) to direct any fluid that does enter the channel away from

278 drops in the adjacent well. The communication channels can be jogged or redirected around 279 barriers (Fig. 3(d)) to, for example, inhibit ballistic motion of fluid during impulsive

280 accelerations.

281 The dimensions, shape and location of these channels determine their effectiveness in

282 allowing vapor communication while inhibiting or preventing liquid transfer.

283 When a microplate with communication channels is tilted from the horizontal so that

284 liquid flows into contact with the communication channel openings on one side of the barrier

285 wall, the hydrostatic pressure in the liquid as well as the pressure associated with surface

286 tension forces will drive any liquid motion through the communication channel. For

287 continuous flow of aqueous solutions in small channels, the Reynold's number is small and

288 the flow is viscous. The volume flow rate is then related to the pressure difference Δρ, the

289 channel radius r (for an approximately circular cross-section channel), the channel length L

290 and the fluid viscosity η by Q =

and the average flow velocity is v_av =

291 Assuming η=8.9 x 10^"4 Pa s (pure water) and typical values appropriate for 96 well SBS

292 standard crystallization microplates of L~ 1 mm, Aphydrostatic ~ pgh ~ 50 Pa (with h = 0.5 cm, a

293 typical well height). With r~75 micrometers (a feature size that can be conveniently injection

294 molded), the average flow velocity is then ~4 cm/s and the flow rate is -0.7 μΐ/s. In the case

295 of a microplate that is accelerated with acceleration a rather than tilted, the maximum

296 Δρ-pah, so for a»g the flow rates can be larger. If the acceleration is transient (e.g., due to a

297 bump), the total flow can be small even if a is large. The viscosity of air, 1.78 x 10^"5 Pa s, is

298 50 times smaller than that of water, so flow rates for a given pressure difference are much

299 larger.

300 A second and, for reducing liquid transfer between wells, more important effect is the

301 pinning of liquid contact lines by solid surfaces. The contact angle Θ formed by the air- liquid

302 interface at a solid surface is determined by the properties of the liquid and surface. In a

303 channel or tube, this leads to the formation of a curved liquid meniscus. For a meniscus with a

304 given contact angle Θ, the pressure difference between the liquid and the air on the other side 305 of the meniscus is Δρ = 2γ cos(0) / r, where γ is the liquid surface tension and r is the radius of

306 the channel or tube. The liquid contact line and the meniscus will remain pinned for some

307 range of values Θ between 0_min and 0_max; the difference between these extreme angles is the

308 contact angle hysteresis, and is determined by the wall roughness, among other factors. This

309 contact angle hysteresis is the analog of static friction for a liquid contact line. Consequently,

310 a minimum pressure in the liquid is required to induce flow through the channel or tube, given

311 by Ap_min = 2γ cos(0_max) / r. Using a typical 0_max -140°, γ=0.0728 N/m (water) and r=75

312 micrometers gives

Pa. Thus, for sufficiently small channels, the pressure

313 difference required to produce flow will be larger than the hydrostatic pressure difference

314 generated when the microplate is tilted or inverted. For 150 micrometer wide channels, it will

315 be roughly 30 times larger, suggesting that microplate accelerations up to -30 g will not cause

316 fluid motion.

317 Experiments have been performed on 96 well SBS standard microplate prototypes

318 with rectangular and trapezoidal cross-section communication channels. Channel dimensions

319 ranged from 0.5 by 0.25 millimeters to 0.25 by 0.075 millimeters. Wells were roughly 4 mm

320 deep. In all cases, even when the reservoir well was completely filled with liquid, no liquid

321 transfer occurred when the microplates were tilted to any orientation, consistent with the

322 above calculations. Communication channels of these dimensions had only small effects on

323 the rate at which vapor pressure equilibration occurred between solutions in wells separated

324 by these channels; this equilibration rate is determined primarily by the rate of evaporation

325 from the liquid surface, which depends on its surface area exposed to air. Since accelerations

326 during mishandling (e.g., dropping the plate) are transient, any liquid volume that is

327 transferred between wells tends to be extremely small. This transfer can be inhibited to some

328 extent by placing a small barrier or "splash guard" in front of the entrance and exit of the

329 communication channels; and by extending the length of the channel by curving, bending or

330 jogging it. Excess pressure developed during accelerations must first drive flow through the

331 entire length of the channel. If the communication channel width is small enough and the path 332 long enough, the liquid will not reach the other well during the duration of the acceleration

333 and associated excess pressure. However, repeated large accelerations may eventually drive

334 very small amounts of fluid out of the communication channel and into the adjacent well.

335 In addition to minimizing liquid transfer volumes, appropriately arranged

336 communication channels can also minimize the effects of small liquid transfers, especially

337 those occurring due to rough handling and impulsive forces. For example, the channels can be

338 arranged so that any liquid that flows into them from the reservoir well and then out of them

339 to the protein/adjacent well is unlikely to contact the protein drops on the bottom of the well.

340 This can be accomplished by directing the communication channel outlets away from the

341 drops. For example, as shown in Fig. 3 (a-c), when the protein drops are disposed away from

342 the ends of the well, the channels can be disposed at either end and directed away from the

343 protein drops. Guards placed at the outlet of the channel can be used to deflect liquid down

344 the sides of the well wall.

345 Vapor communication channels can also provide some control over the rate of vapor

346 transfer and vapor equilibration between connected wells. The rate of transfer of volatile

347 components of the liquids in the wells - including water and alcohols - depends upon the rate

348 of evaporation per unit area from the liquid-air interface, the surface area of liquid-air

349 interface, and the rate of vapor diffusion and convection. The communication channels affect

350 vapor diffusion, by constricting the area through which diffusion occurs, and vapor

351 convection, since convection within the channels is strongly suppressed in sufficiently small

352 channels, e.g., those of the prototypes described above. The modulation of the net rate of

353 transfer of volatile components between wells is determined by which process - evaporation

354 from the liquid surface, convective and diffusive transport within each well, or diffusive

355 transport through the channel - is slowest. For highly volatile components like alcohols, the

356 effects of the channels may be dominant, but for slowly evaporating solutions like aqueous

357 buffer containing 30% polyethylene glycol, the evaporation rate may be limiting. In any case,

358 reducing the channel cross-section dimensions and increasing their length should eventually 359 make transport through the channel the limiting step. The channel dimensions and the total

360 number of channels can then be used to control - specifically, to reduce relative to the large-

361 area channel limit - the rate of vapor transport and equilibration between wells. Filling the

362 channels with an oil or other non-volatile material could be used to further reduce diffusion

363 through the channel and thus to further reduce the rate of vapor equilibration. In protein

364 crystallization, slower equilibration is often desirable, as it can produce less nucleation and

365 larger crystals.

366 Liquid retention ledge/apertures

367 As shown in Figure 4(a)-(d), in its simplest form, the liquid retention ledge/aperture is

368 formed by a ledge 55 that projects outward from the walls 45 of the well, at some position

369 between the bottom and top of the well. The parameters of this ledge are its thickness, the

370 ratio of this thickness to the well depth, the dimensions of its inner aperture 60 (or the amount

371 by which the ledge projects outward from the well wall) and its height above the bottom of

372 the well. The ledge can be tilted upward or downward. It may have a rectangular cross-section

373 (Fig. 4(a) and (b)). It may instead have a bottom surface that, e.g., curves to meet with the

374 wall (Fig. 4(c) and (d)). It may have projecting or raised lips immediately adjacent to the

375 aperture to control contact line pinning there. There may be more than one aperture in a given

376 well. The shape and size of the aperture formed by the ledge should be optimized to prevent

377 liquid motion through the aperture when the plate is tilted, inverted or roughly handled, and

378 should also allow easy filling using standard liquid handlers and pipetters. The well bottoms

379 may be open - to be sealed by a separate part - as in Fig. 4(b) and (d); or one or all wells

380 within a cell may have an enclosed bottom, as in Fig. 4 (e-g). The wells within a cell may

381 have the same depth (Fig. 4(b) and (d)), or they may have different depths (Fig. 4(e-g)).

382 Current 96 cell microplates for protein crystallization have reservoir well volumes of

383 roughly 40 to 200 microliters. Since most plates conform to the SBS dimensions, well

384 volumes decrease with the number of cells in the plate. With low-water vapor permeability

385 plastics such as cyclic olefin copolymer (COC), well volumes can be reduced to 386 approximately 10 microliters for experiments lasting one month, and to smaller volumes for

387 shorter experiments, without appreciable effects due to water loss from the cells.

388 The plastics used in conventional injection molded microplates tend to be somewhat

389 hydrophobic. As illustrated in Fig. 5(a), when aqueous solutions are dispensed into

390 conventional flat or curved bottom wells, they tend to form hemispherical domes 90 on the

391 bottom of the well 75 rather than wet and spread uniformly throughout the well. The liquid

392 may reside only near the center of the typically rectangular or elongated wells, or it may be

393 drawn to a corner. Consequently, for fixed volume the exposed surface area of the liquid will

394 vary, changing the rate of equilibration of the liquids in each well. This also makes it more

395 difficult to completely fill a well without it overflowing, or without having liquid touch the

396 top sealing film 85 when the plate is sealed. Figure 6(a) shows a sequence of images acquired

397 during filling of the reservoir well of a commercial protein crystallization microplate. As

398 liquid is added, the liquid often accumulates in one side of the well, and the liquid level rises

399 to the top of the well on that side, before the liquid finally spreads across the entire bottom of

400 the well. Fully filling the well is usually impossible without having liquid contact the top

401 sealing surface, which can prevent plate sealing. If the sealing film is not applied, when the

402 plate is tilted to 90 degrees or inverted, most or all of the liquid will flow out of the well.

403 With the top sealing film applied, tilting or inversion will typically result in substantial liquid

404 transfer between wells.

405 As illustrated in Fig. 5(b), by adding the liquid retention ledge 125 and aperture 115,

406 as liquid is injected into the well, the liquid's top surface 110 eventually contacts and wets the

407 bottom surface of the retention ledge. The liquid then spreads laterally (120) beneath the ledge

408 as additional liquid is added. Surface tension forces prevent spreading of the liquid through

409 and out the aperture, so the liquid will then expand laterally beneath the ledge until the

410 volume beneath the ledge is nearly completely filled (arrows).

411 The minimum outward projection of the ledges from the well wall required to keep

412 liquid from rising to the top surface of the plate (and contacting the sealing film) depends on 413 the shape of the liquid drop formed on the bottom of the well during filling, on the ratio of the

414 well depth to well width, on the vertical position of the ledge relative to the bottom of the

415 well, and also on how the plate is filled. The drop shape depends on its volume, surface

416 tension and contact angle at the well bottom. For a small depth to width ratio and/or for a

417 ledge placement near the top of the well as in Fig. 5(b), the ledge must be relatively wide,

418 whereas for a large depth to width ratio or for ledge placement closer to the bottom of the

419 well, the ledge can be relatively small. For rectangular or otherwise elongated wells, the ledge

420 width can be reduced at the narrower ends of the wells relative to on the wider sides of the

421 wells, as the ledges on the wider sides will largely determine liquid retention. If the liquid is

422 not dispensed in the center of the well but is dispensed toward one side, the ledge projection

423 can be reduced and still effectively contain the liquid.

424 Experiments using prototype 96 cell microplates have confirmed that liquid retention

425 ledges cause liquid spreading and more uniform well filling for water and variety of aqueous

426 solutions and mixtures ("screens") containing alcohols, ethylene and polyethylene glycols,

427 glycerol, salts, detergents and other organic compounds commonly used in protein

428 crystallization. Fig. 6(a) shows a sequence of images as liquid is added to a conventional well

429 on a commercial 96 cell plate. The liquid spreads and then rises to the top surface of the plate

430 at the left end of the plate (so that it will contact the sealing film when the plate is sealed)

431 before continuing to spread to the right end. When the plate is tilted, the liquid freely flows

432 out of the well. Figure 6(b) shows a corresponding sequence of images in a prototype plate

433 according to the present invention. As liquid is added, the liquid quickly contacts the bottom

434 surface of the ledge, and then spreads uniformly beneath the ledge until the entire volume

435 below the ledge (with the possible exception of a small bubble) is filled. Even with the

436 sealing film not applied, the plate can be tilted to any orientation or inverted without liquid

437 flow out of the well through the aperture. Using the well dimensions of Fig. 6(b),

438 experiments with ledges of varying widths / extensions from the well sidewall suggest that the

439 utility of the ledges in promoting liquid spreading and more uniform well filling is largely

440 insensitive to ledge width, for widths of at least 0.2 mm. 441 When the microplate is tilted or inverted, the liquid retention ledge/aperture inhibits

442 liquid flow out though the aperture by several mechanisms. First, as shown in Fig. 7, as the

443 liquid 165 begins to bulge through the aperture formed by ledge 160, it must increase its

444 surface area and so there is surface-tension-related pressure that opposes this bulging. This

445 opposing pressure increases with the amount of bulge to a maximum given roughly by

446 cos0_max /r where γ is the surface tension of the liquid-air interface, r is the aperture

447 radius, and 0_max is the maximum stable contact angle of the liquid-air interface relative to the

448 top surface of the aperture. This pressure (roughly 150 Pa for water in a 1 mm radius

449 aperture), can exceed the hydrostatic pressure in the liquid in a shallow well, and prevent it

450 from moving. In order to obtain maximum benefit from this effect, the top sealing surface

451 155 of the microplate must be far enough away so that liquid bulging through the aperture

452 does not contact the sealing film sealing this top surface when, e.g., the plate is inverted after

453 filling.

454 Second, the liquid is incompressible. Thus, as the liquid displaces into and then bulges

455 out of the aperture, the gas volume in the unfilled space 170 on the "filled" side of the

456 aperture must increase, producing a decrease in pressure in that space that opposes the

457 motion. If the initial gas volume is very small, the pressure drop for even small displacements

458 may be large. For example, if the unfilled volume below the aperture in a 40 microliter well is

459 4 microliters (10%), the formation of a 1 millimeter diameter hemispherical liquid bulge

460 through the aperture produces a pressure drop of 6500 Pa, or roughly 130 times larger than

461 hydrostatic pressure.

462 Several design features can increase the filled volume fraction occupied by the

463 incompressible liquid below the liquid retention ring and minimize the unfilled volume

464 fraction occupied by compressible air. The well can be made more nearly square or circular,

465 as viewed from above. The corners 70 of the well can be rounded (Fig. 4(c) and (d)) to reduce

466 trapping of air bubbles there, especially below the bottom surface of the ledge. 467 Wider ledges tend to trap air beneath the ledge at either end of a rectangular well. This

468 trapping can be reduced by reducing the width of the ledge. For rectangular or otherwise

469 elongated wells, the width can be reduced only at the well ends (Fig. 8(b)), minimizing air

470 trapping while minimizing the width of the aperture. Small diameter (e.g., 150 micrometer)

471 "vent" apertures can be disposed in the ledge at either end of the well, allowing air to escape

472 through them during filling from the center. The ledge can be then be extended to form a

473 continuous sheet across the well, with holes for filling and venting (Fig. 8(c)). The filling

474 aperture can be disposed at one end of the well and a single vent aperture at the other end

475 (Fig. 8(d)). The filling aperture can be disposed in the middle, and thin slits extended from

476 either side of it as in Fig. 8(e) to allow gas escape during filling but to minimize fluid motion

477 out compared with, e.g., the apertures in Fig. 8(a) and (b) during handling and acceleration.

478 Third, in order for liquid to flow out of the aperture during, e.g., microplate rotation to a

479 vertical or inverted orientation, air must flow in through it. If the well below the aperture is

480 filled, the aperture will remain covered by liquid during rotation, so an air bubble must form

481 and move through the aperture in order for liquid to flow out. Because of the large pressure

482 increase that inhibits uniform liquid motion out of the aperture, this may involve creating an

483 outward-protruding bubble in part of the aperture and an inward-protruding bubble in another

484 part. The surface-tension-related pressures required to create these bubbles are inversely

485 proportional to the bubble diameter. Consequently, any tendency for these bubbles to form

486 can be suppressed by making the aperture as small as is feasible in all its dimensions. The

487 smallest dimension is limited by the size of the dispensing tips in commercial liquid handlers

488 and pipetters, and by the precision and accuracy in positioning the tips relative to the plate. In

489 Fig. 8(c), a central aperture is provided for liquid filling and two much smaller vent apertures

490 at either end of the well allow air escape during filling.

491 Finally, any liquid motion and especially the bubble formation described above occur

492 on a timescale that is determined by the liquid's viscosity. This viscosity thus inhibits bubble

493 formation and liquid flow out of the aperture in response to impulsive and other short-

494 duration perturbations. 495 Experiments on microplate prototypes with 3-4 mm deep reservoir wells and liquid

496 retention ledges/apertures filled with volumes between 20 and 40 microliters show that the

497 ledges/apertures prevent all liquid motion out of the well during ordinary manual plate

498 handling, during rotations and inversions, and when the microplate is held for extended

499 periods in vertical and inverted orientations, including when there is no top film sealing the

500 well. This contrasts with the behavior of liquid in prototype wells without ledges (as are used

501 in current microplates), where liquid easily flows out of the reservoir and mixes when the

502 plate is tilted or inverted. Liquid only flows out through the aperture when the plate

503 experiences intense impulsive forces, e.g., those that occur if the plate is dropped from a

504 height of, e.g., two feet onto a hard surface

505 In our experiments, we have used wells with liquid retention ledges that produce

506 apertures of width 2 mm - determined by the ~1 mm typical size of liquid dispensing pipette

507 tips and tolerances in microplate positioning relative to the dispensing tip. Experiments using

508 ledges placed at varying distances below the top surface of the microplate showed that a

509 distance of approximately 0.5 mm gave good results with no tendency for liquid to contact

510 and spread on the top sealing surface for wells 3-4 mm deep. Smaller apertures can be used to

511 increase inhibition of flow during acceleration, but place constraints on the kinds of liquid

512 dispensing tips that can be used to fill the plate and the accuracy of the plate positioning

513 relative to the dispensing tips. Injection molding the liquid retention ledge is difficult if the

514 bottom of the well is also injection molded in the same step - as is the case with all

515 commercial microplates currently sold for protein crystallization. A simpler approach is to

516 seal the well bottom after molding with a separate plastic film or plate or molded part.

517 Molding and release from the mold is then straightforward, lowering cost.

518 Plates combining vapor communication channels and liquid retention ledges.

519 Experiments on 96 well plate prototypes with liquid retention ledges/apertures and 150

520 micrometer wide communication channels (with cells as shown in Fig. 4(b)) show that, when

521 the reservoirs filled with roughly 40 microliters of water (high surface tension) or 30% 522 isopropanol in water (low surface tension) are placed in a single standard bubble bag, they can

523 be dropped from a height of six feet onto a concrete floor with little transfer of liquid between

524 the reservoir wells and the protein drop wells.

525 Microplate storage and shipping assembly/system.

526 In some applications, it is desirable to be able to ship microplates between laboratories

527 by conventional mail or courier service. For example, it is desirable to ship plates from

528 university or industrial laboratories to synchrotron X-ray sources for X-ray inspection. This

529 shipping requires that (1) variations in average temperature be minimized to prevent freezing,

530 precipitation and other effects that may damage the samples in the plate; (2) temperature

531 gradients across the plate be minimized to prevent evaporation and condensation; and (3) peak

532 accelerations be minimized to minimize liquid transfer between wells. (1) and (2) can be

533 achieved using standard commercial shipping containers, such as those comprised of

534 insulating Styrofoam boxes with high heat capacity gel packs inside to maintain temperature.

535 (3) can be achieved using bubble wrap, air pillows, foam or other materials that compress

536 during impact and that have an inelastic (dissipative) response to compression, so as to reduce

537 peak accelerations and thus peak forces exerted on objects contained within them. A

538 commercial microplate system for, e.g., protein crystallization, shipping and X-ray inspection

539 may then comprise a microplate with liquid retention ledges/apertures and narrow vapor

540 communication channels; a cardboard box lined with shock-absorbing foam; an interior

541 thermally insulating Styrofoam container; high heat capacity gel packs; and additional foam

542 or bubble packaging to further reduce accelerations of plates inside the Styrofoam container.

543 During shipping, microplates may also experience reduced ambient pressures.

544 Microplate prototypes with vapor communication channels and liquid retention ledges, and

545 that had both top and bottom surfaces sealed using separate polymer films, were tested in a

546 chamber with an air pressure of 22.2 inches of Hg (75 kPa or roughly 3/4 of sea level

547 atmospheric pressure). This pressure corresponds to the minimum pressure in the cargo hold 548 of a commercial airliner. These tests showed no transfer of liquid between wells or other

549 problems after repeated cycling between reduced pressure and atmospheric pressure.

550 In standard use, the plate bottom is sealed, either during injection molding or by

551 attachment of a separate film or plate or injection molded part. Reservoir liquid is dispensed

552 using a manual pipetter or automated liquid handler through the liquid retaining aperture and

553 into the well below, filling the well below the liquid retention ledge as completely as possible

554 Protein solution is then dispensed as drops on the bottom surface of the adjacent protein well.

555 The top surface of the microplate is then sealed using a vapor-impermeable sealing film. The

556 plate is then rotated to a desired orientation (usually either its original horizontal orientation

557 or an inverted orientation) for crystal growth. The plate is then rotated to another orientation

558 (usually vertical) to examine the contents of each cell using X-rays, UV or visible light, or

559 other probes. The plate may also be rotated to a vertical orientation immediately after filling,

560 allowing vertical storage and inspection during the experiment. This may be preferable to

561 storage in a horizontal orientation if a plate to be inspected in a vertical orientation multiple

562 times during an experiment.

563 The description above has presented several alternative embodiments. The invention is

564 not restricted to microplates for protein crystallization, or to microplates. It can be used in any

565 kind of microplate. It can also be used in any kind of device where it is desirable to have

566 vapor communication between two or more wells or chambers and easy filling and removal of

567 material from wells from a top surface, while preventing liquid transfer between wells during

568 microplate rotation or acceleration, and allowing device use in any orientation.

569 All references, including publications, patent applications, and patents cited herein are

570 hereby incorporated by reference in their entireties to the extent allowed, and as if each

571 reference was individually and specifically indicated to be incorporated by reference and was

572 set forth in its entirety herein. 573 The use of the terms "a" and "an" and "the" and similar referents in the context of

574 describing the invention (especially in the context of the following claims) is to be construed

575 to cover both the singular and the plural, unless otherwise indicated herein or clearly

576 contradicted by context. The terms "comprising," "having," "including," and "containing" are

577 to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless

578 otherwise noted. The term "connected" is to be construed as partly or wholly contained

579 within, attached to, or joined together, even if there is something intervening.

580 The recitation of ranges of values herein is merely intended to serve as a shorthand

581 method of referring individually to each separate value falling within the range, unless

582 otherwise indicated herein, and each separate value is incorporated into the specification as if

583 it was individually recited herein.

584 All methods described herein may be performed in any suitable order unless otherwise

585 indicated herein or otherwise clearly contradicted by context. The use of any and all

586 examples, or exemplary language (e.g., "such as") provided herein, is intended merely to

587 better illuminate embodiments of the invention and does not impose a limitation on the scope

588 of the invention unless otherwise claimed.

589 No language in the specification should be construed as indicating any non-claimed

590 element as essential to the practice of the invention.

591 It will be apparent to those skilled in the art that various modifications and variations

592 can be made to the present invention without departing from the spirit and scope of the

593 invention. There is no intention to limit the invention to the specific form or forms disclosed,

594 but on the contrary, the intention is to cover all modifications, alternative constructions, and

595 equivalents falling within the spirit and scope of the invention, as defined in the appended

596 claims. Thus, it is intended that the present invention cover the modifications and variations

597 of this invention provided they come within the scope of the appended claims and their

598 equivalents.

Claims

600 What is claimed is:

601 1. A microplate comprising:

602 a frame comprising a substantially planar top surface, a bottom surface, and including

603 a plurality of cells formed therein, each cell comprising:

604 a first well;

605 a second well;

606 wherein said first well and said second well are separated by a separation wall

607 extending in a first direction from the bottom surface to the top surface of said frame,

608 and extending in a second direction along a longitudinal axis; and

609 wherein said first well and said second well are connected via at least one

610 vapor communication channel structured to allow vapor communication between said

611 first well and said second well, and to facilitate the inhibition of liquid transfer

612 between said first well and said second well when at least one cell contains a liquid

613 and when said frame is tilted from a horizontal position or when said frame is

614 subjected to impulsive accelerations.

615 2. The microplate of claim 1, wherein one of said first well and said second well has a

616 volume of between about 10 microliters to about 200 microliters.

617 3. The microplate of claim 1, wherein said at least one vapor communication channel is

618 carved into the top surface of said frame.

619 4. The microplate of claim 3, wherein a top surface of said separation wall comprises a

620 center point and a two end points, and wherein said at least one vapor communication channel 621 is carved into the top surface of said separation wall at a position between the center point and

622 one of the two end points of the top surface of said separation wall.

623 5. The microplate of claim 4, wherein said at least one vapor communication channel is

624 located adjacent to one of the two end points of the top surface of said separation wall.

625 6. The microplate of claim 4, wherein said at least one vapor communication channel is

626 carved into the top surface of said separation wall at an angle to the longitudinal axis.

627 7. The microplate of claim 3, wherein said at least one vapor communication channel is

628 non-linear.

629 8. The microplate of claim 7, wherein said at least one vapor communication channel is

630 carved into the top surface of said separation wall in a jogged manner.

631 9. The microplate of claim 3, wherein said at least one vapor communication channel has

632 a width of between about 0.075 millimeters to about 0.25 millimeters.

633 10. The microplate of claim 1, further comprising a plurality of vapor communication

634 channels structured to allow vapor communication between said first well and said second

635 well, and to facilitate the inhibition of liquid transfer between said first well and said second

636 well when at least one cell contains a liquid and when said frame is tilted from a horizontal

637 position or when said frame is subjected to impulsive accelerations, wherein each of which of

638 said plurality of vapor communication channels is carved into the top surface of said frame.

639 11. The microplate of claim 1, further comprising a liquid retention ledge that is

640 connected to and extends around at least a portion of an interior perimeter of one of said first

641 well and said second well at a predetermined distance below the top surface of the frame, and 642 projects outward from the interior perimeter forming an aperture with a diameter that is

643 smaller than a diameter of the interior of said one of said first well and said second well.

644 12. The microplate of claim 11, wherein said liquid retention ledge is structured to

645 facilitate uniform filling of said one of said first well and said second well with liquid as

646 liquid is added and to facilitate the prevention of the formation of air bubbles therein.

647 13. The microplate of claim 12, wherein said liquid retention ledge is structured to pin the

648 liquid contact line when liquid is added to said one of said first well and said second well

649 thereby facilitating the prevention of the liquid from rising above the level of the liquid

650 retention ridge during the addition of the liquid.

651 14. The microplate of claim 13, wherein said liquid retention ledge projects outward at a

652 distance of at least 0.2 millimeters from the interior perimeter of said one of said first well and

653 said second well.

654 15. The microplate of claim 11, wherein said liquid retention ledge is structured to

655 facilitate the prevention of liquid from flowing out of said one of said first well and said

656 second well when said frame is tilted from a horizontal position or when said frame is

657 subjected to impulsive accelerations.

658 16. The microplate of claim 15, wherein said liquid retention ledge comprises a

659 rectangular cross-section.

660 17. The microplate of claim 15, wherein said liquid retention ledge comprises a curved

661 bottom portion, wherein said curved bottom portion of said liquid retention ledge is structured 662 to facilitate the prevention of air bubble trapping during filling of said one of said first well

663 and said second well with liquid. 664

665 18. The microplate of claim 17, wherein said liquid retention ledge is structured to form a

666 plurality of apertures.

667 19. The microplate of claim 18, wherein a first one of said plurality of apertures is formed

668 adjacent to the interior perimeter of said one of said first well and said second well.

669 20. The microplate of claim 19, wherein a second one of said plurality of apertures is

670 substantially centrally formed in said one of said first well and said second well.

671 21. The microplate of claim 20, wherein each of said first and said second one of said

672 plurality of apertures is substantially circle- shaped.

673 22. The microplate of claim 21, wherein a diameter of said second one of said plurality of

674 apertures is larger than the diameter of said first one of said plurality of apertures.

675 23. The microplate of claim 22, wherein said first one of said plurality of apertures has a

676 diameter of about 150 micrometers.

677 24. The microplate of claim 11, wherein said one of said first well and said second well

678 including said liquid retention ledge further comprises a bottom portion with rounded corners.

679 25. A microplate comprising:

680 a frame comprising a substantially planar top surface, a bottom surface, and including

681 a plurality of cells formed therein, each cell comprising:

682 a first well;

683 a second well; 684 wherein said first well and said second well are separated by a separation wall

685 extending in a first direction from the bottom surface to the top surface of said frame,

686 and extending in a second direction along a longitudinal axis; and

687 a liquid retention ledge that is connected to and extends around at least a

688 portion of an interior perimeter of one of said first well and said second well at a

689 predetermined distance below the top surface of the frame, and projects outward from

690 the interior perimeter forming an aperture with a diameter that is smaller than a

691 diameter of the interior of said one of said first well and said second well, wherein said

692 liquid retention ledge is structured to facilitate uniform filling of said one of said first

693 well and said second well with liquid as liquid is added and to facilitate the prevention

694 of the formation of air bubbles therein.

695 26. A microplate comprising:

696 a frame comprising a substantially planar top surface, a bottom surface, and including

697 a plurality of cells formed therein, each cell comprising:

698 a first well;

699 a second well;

700 wherein said first well and said second well are separated by a separation wall

701 extending in a first direction from the bottom surface to the top surface of said frame,

702 and extending in a second direction along a longitudinal axis; and

703 a liquid retention ledge that is connected to and extends around at least a

704 portion of an interior perimeter of one of said first well and said second well at a 705 predetermined distance below the top surface of the frame, and projects outward from

706 the interior perimeter forming an aperture with a diameter that is smaller than a

707 diameter of the interior of said one of said first well and said second well, wherein said

708 liquid retention ledge is structured to facilitate the prevention of liquid from flowing

709 out of said one of said first well and said second well when said frame is tilted from a

710 horizontal position or when said frame is subjected to impulsive accelerations.

711