Abstract:
An apparatus and methods for performing microchemical manipulation and analysis of liquid samples. One or more droplets of liquid are loaded serially or in parallel onto a flexible member which may be a fiber or a tape, and are retained on the flexible member by virtue of surface adhesion. Microchemical operations may be performed on these droplets, the operations including mixing, dilution, concentration, heating, cooling, filtering, and analyzing, where the analyzing may include chemical, biochemical, optical, or other physical analyses.

Description:
This application claims benefit of U.S. provisional application No. 60/057,734, filed Aug. 28, 1997, which is herein incorporated by reference. 
    
    
     TECHNICAL FIELD 
     The present invention pertains to an apparatus and methods for transporting a massive number of droplets of liquid and for performing microchemical operations on these droplets, the operations including mixing, dilution, concentration, heating, cooling, filtering, and analyzing, where the analyzing may include chemical, biochemical, optical, or other physical analyses. 
     BACKGROUND OF THE INVENTION 
     Chemistry on the micro-scale, involving the reaction and subsequent analysis of quantities of reagents or analytes of order microliters or smaller, is an increasingly important aspect of the development of new substances in the pharmaceutical and other industries (e.g., synthesis and analysis of new conductive polymers, phosphors, superconductors, etc.). Such reaction and analysis must accommodate vast libraries of compounds to be reacted and analyzed under various conditions. Significant problems associated with current technologies as applied to chemical analysis of vast numbers (potentially on the order of hundreds of thousands or millions per day) of compounds include the problem of conveying analytes from well plates in which libraries of compounds are kept to regions of reaction and analysis, potential contamination of the transport medium, and the sheer size of the facility required to handle vast numbers of compounds and reactions. 
     Existing technology applies 96-well plates containing quantities on the order of 1 milliliter of liquid compound per well, and, generally, envisions chemical reactions and analysis on flat, two-dimensional surfaces such as silicon chips. In addition to the parallel processing of liquid samples provided by chip technology, a method is required to perform serial steps of liquid sampling, transport, and microchemical analysis. 
     SUMMARY OF THE INVENTION 
     In accordance with a preferred embodiment of the invention, there is provided an apparatus for transporting one or more droplets of a liquid. The apparatus has a reservoir for containing a quantity of the liquid and a flexible member for receiving the droplet or droplets of liquid from the reservoir, with the droplets adhering to the flexible member by virtue of the surface adhesion of the liquid. The flexible member may be a fiber or a ribbon, and a portion of the flexible member may be formed at least temporarily into an enclosure for encapsulating at least one droplet of the liquid. 
     In accordance with a preferred embodiment of another aspect of the present invention, there is provided an apparatus for storing or analyzing a plurality of liquid droplets. The apparatus has a drum having a substantially cylindrical surface and a plurality of wells disposed within the substantially cylindrical surface. The apparatus also has a dispenser for injecting the plurality of droplets into the wells according to a predetermined schedule and may have an analyzer for characterizing at least one chemical property of the droplets. The dispenser may be a flexible transport member wrapped onto the drum in such a manner as to position no more than one droplet of liquid in each well of the drum. 
     In accordance with other aspects of the present invention, methods are provided for transporting, sampling, mixing, concentrating, diluting, heating, cooling, and analyzing liquid samples by extracting a volume of the liquid by means of the apparatus discussed above. Analysis of physical and chemical properties of the samples may be performed in series or in substantial parallelism. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The invention will be more readily understood by reference to the following description, taken with the accompanying drawings, in which: 
     FIGS. 1A-1C are side views in cross-section of a droplet in adhesion to a fiber in accordance with an embodiment of the present invention; 
     FIG. 2A is a side view in cross section of a liquid take-up mechanism in accordance with an embodiment of the present invention; 
     FIGS. 2B-2D show successive steps of liquid take-up onto a fiber in accordance with the embodiment of FIG. 2A; 
     FIG. 3 depicts the transport of multiple droplets on a fiber, and an advancement mechanism for translating the fiber in accordance with an embodiment of the invention; 
     FIG. 4 depicts the combination of droplets transported to the cite of combination by respective fibers in accordance with an embodiment of the present invention; 
     FIG. 5 depicts the respective surface areas and volumes of individual and combined droplets borne by a fiber in accordance with an embodiment of the present invention; 
     FIG. 6 shows the transport and combination of droplets of varying sizes in accordance with an embodiment of the invention; 
     FIG. 7 is a schematic diagram of a capacitive monitor for sensing the position and characteristics of a microdroplet in accordance with an embodiment of the invention; 
     FIG. 8 is a plot of the measured capacitance as a droplet traverses the capacitive monitor of FIG. 7; 
     FIG. 9 is a schematic diagram of a typical optical configuration employed for interrogating a droplet in accordance with an embodiment of the present invention; 
     FIG. 10 is a perspective view of a sample handling drum for storage and analysis of microsamples conveyed by fiber in accordance with embodiments of the invention; 
     FIG. 11A is a side view in cross section of a liquid take-up mechanism employing ribbon or tape in accordance with an alternate embodiment of the present invention; 
     FIGS. 11B-11C show cross-sections of pulleys employed to impart curvature to the ribbon or tape of FIG. 11A; and 
     FIG. 12 is a perspective view of a ribbon employed in curled and flattened configurations for transport of microdroplets in accordance with embodiments of the invention. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Referring first to FIG. 1A, it is recognized that transport, manipulation, and microchemical reaction of materials in the liquid phase or in liquid suspension need not be conducted in a planar environment but may use the adhesion of a droplet  10  to a fiber  12  in order to achieve one or more of the enumerated processes. Processes of adhesion of a liquid to the surface of a solid are discussed, for example, in D. Myers,  Surfaces, Interfaces, and Colloids: Principles and Applications , (VCH Publishing, N.Y. 1991), which is incorporated herein by reference. Chapter 17, entitled “Wetting and Spreading,” at pp. 349ff, is of particular relevance. 
     “Fiber,” as used in the present description and in any appended claims, refers to any flexible material the linear dimension of which substantially exceeds the transverse dimensions of the material and includes materials which may be amorphous, such as glass or plastic. It is the geometry rather than the composition of the fiber which is germane to the overall scope of the invention, although the properties of different compositions of fiber may be advantageously exploited, as further described below. The transverse section of the fiber may have any specified shape, thus the fiber may be of circular or elliptical cross section, in the manner of optical fibers, for example. The flexible member may also be flat and ribbon-like, or curled, as discussed below, and references herein to “fiber” are to be understood as also encompassing the generality of flexible members. Materials may include quartz or glass, for example, or amorphous metal (metallic glass), as another example. Metal wires of diameters as small as 25 μm, or even 5 μm, are readily available. Droplet  10  may be referred to herein and in the appended claims as a “microdroplet” or a “sample,” and may include droplets containing living cells, such as yeast cells, for example and may include droplets carrying a single living cell per droplet. 
     The fiber system described herein for droplet microchemistry may entirely avoid the use of tubes which require cleaning, and may overcome various handling difficulties associated with samples enclosed in conduits. By way of contrast, glass fibers are available at low cost in lengths as long as many kilometers and are readily disposed of after chemical manipulation and analysis are completed. 
     The following components of a microchemical analysis system are examples of processes which may be carried out through the use of fiber technology in accordance with various embodiments of the present invention: 
     1. Sampling—i.e., transferring a small amount of a specified fluid to a fiber from either a reservoir of fluid or another surface; 
     2. Transporting fluid samples; 
     3. Adding one sample to another; 
     4. Diluting a sample; 
     5. Concentrating a sample; 
     6. Heating or Cooling a sample; 
     7. Extracting or filtering chemicals contained within a sample; 
     8. Analyzing chemicals as to their composition or other chemical or physical characteristics. 
     The quantity of fluid adhering to fiber  12  and thus constituting droplet  10  is a function of the respective compositions of fiber  12  and droplet  10  and of the method in which adhesion of the droplet to the fiber is attained. In practice, variations in volume of over a factor of 1000 have been achieved. The surface tension of droplet  10 , for example, serves to counter unlimited diffusion of the fluid along the length of the fiber. Where fiber  12  is not wetted by droplet  10 , tangent  16  to outer surface  14  of droplet  10  forms contact angle  18  with the local surface of fiber  12 . Where wetting of fiber  12  by droplet  10  occurs, as shown in FIG. 1B, the spread of droplet  10  along fiber  12  is limited by surface tension. The size of the droplet is related to the transverse dimension (or dimensions) of the fiber; for example, a fiber of 100 μm diameter may hold a droplet of over five times that diameter. 
     For transportation of droplet  10 , it is preferred that droplet  10  surround fiber  12 , as shown in FIG.  1 C. 
     Sampling of materials in liquid form, thereby loading the fiber, is now described with reference to FIG.  2 . Fiber  12  may be guided, by any mechanical arrangement known in the mechanical arts, so as to come in contact with liquid  18  which may be contained within reservoir  20 . Quartz is a preferred material for fiber  12  as it is very strong. Fiber  12  may be guided, for example, by means of pulleys  22 , comprising pulley feed mechanism  23 , so as to form loop  24 . Loop  24  is brought in a direction designated by arrow  26  into contact with liquid  18 , as shown in FIG. 2B, preferably without penetrating the surface of the liquid. As loop  24  is withdrawn in direction  28  away from the surface of the liquid, as shown in FIG. 2C, droplet  10  begins to form on fiber  12 . As loop  24  is withdrawn further, droplet  10  detaches from liquid reservoir  20 , as shown in FIG.  2 D. 
     The rate at which fiber  12  is withdrawn from liquid reservoir  20  contributes to determining the size of droplet  10  that is formed, with a slower withdrawal resulting in a smaller droplet. Higher velocities result in larger droplets, whereas higher velocities yet result in loss of the droplet to dripping back into the reservoir. Pulley feed mechanism  23  also serves to impel fiber  12  in the direction of its longitudinal axis. 
     A typical volume of fluid sampled by a fiber in the manner described is of the following order: assuming a sphere of fluid having a diameter of 300 μm surrounding a fiber of 100 μm diameter, and removing the volume displaced by the presence of the fiber, the volume of the liquid droplet is on the order of 12 nanoliters. Since the volume of the droplet scales substantially as the cube of the characteristic transverse dimension of the fiber, a fiber of 1 mm diameter may be used to transport droplets of volume on the order of 10 microliters. In fact, droplet volumes between 10 picoliters and 10 microliters are readily handled in accordance with the invention. 
     Referring, once more, to FIG. 2A, contact between fiber  12  and the liquid in reservoir  20  may be achieved in any of a variety of ways. In accordance with a preferred embodiment of the invention, reservoir  20  may be raised so that surface  26  of liquid  18  makes contact with fiber loop  24 .Alternatively, the entire pulley feed mechanism  23  may be lowered to that loop  24  makes contact with liquid  18 , or, in accordance with another embodiment, the speed of rotation of pulleys  22  may be differentially governed to regulate the length of loop  24 . It is to be understood that additional pulleys may be provided within the scope of the invention, such as to provide for differential advancement of the leading and trailing knees of loop  24 . 
     Transportation 
     A fiber, as described, may hold a large number of liquid droplets and convey all the droplets in a specified direction for serial handling on a massive scale. Referring now to FIG.  3  and considering fiber  12  to have a diameter of 100 μm, for example, droplets  10  may be conveyed with an inter-droplet spacing  30  of 500 μm or less. Thus, over 2000 droplets may be loaded per meter of fiber, and 500 m of fiber can hold and transport over one million droplets, where the composition of the individual droplets may vary. 
     Additionally, in accordance with a further embodiment of the invention, small solid particles  31  may be held to fiber  12  by electrostatic attraction or by forces of liquid surface adhesion and similarly transported, manipulated and analyzed by the disclosed system. In order for fiber  12  to be advanced by pulleys  22 , provision must be made not to interfere with the conveyance of droplets  10 . One method of providing for fiber advancement is to provide radial members  32  that contact fiber  12  only at positions  34  where no droplets are present. Pulleys  22  are shown in this figure to be counterrotating so as to advance fiber  12  in direction  36 , to the right in this figure. 
     In an alternate embodiment of the invention, the fiber may be magnetized at specified locations or intervals and a magnetically attractable bead may be attracted at those locations and surrounded by the liquid to be transported. 
     Once droplets  10  have been loaded onto fiber  12 , they may be heated or cooled, and functions of microchemistry and analysis may be performed on them, as discussed in greater detail in the following discussion. 
     Addition of Chemicals Via Fiber Technology 
     Referring now to FIG. 4, distinct droplets, of the same or different chemical composition, may be combined using the droplet microchemistry methods of the present invention. A first fiber  40 , carrying droplets  42  may be moved with respect to a second fiber  44  carrying other droplets  46  such that fibers  40  and  44  lie in substantially locally parallel planes and have a specified relative angle  48  which may be a right angle. When fiber  40  is brought within sufficient proximity of fiber  44 , a single droplet  49  may be formed by the combination of droplets from each of fibers  40  and  44 . When fiber  40  is now moved away from fiber  44 , combined droplet  49  tends to move to one or the other of fibers  40  and  44 . In order to select which fiber will receive combined droplet  49 , one fiber, say, for example, fiber  44 , may be moved substantially transversely to the other fiber, and then the fibers are separated. The fibers are distinguished in this way since the force required to move a droplet along a fiber differs from the force required to remove the droplet from the fiber or to impart momentum to the liquid in a direction tangential to the circumference of the fiber. 
     In the manner described, thousands of droplets conveyed by a single fiber may be combined with thousands of droplets conveyed by a second fiber by sequentially bringing the droplets into mutual contact. 
     In FIG. 5, the surface areas and volumes of two droplets  50 , assumed to be equal, are compared with the same quantities of combined droplet  52  which results from the combination of the original droplets  50  in accordance with the preceding discussion. While the volume of fluid of droplet  52  is twice that of either droplet  50 , the surface area of droplet  52  is less than twice that of either droplet  50 . Since the surface area of droplet  52 , and thus its surface energy which increases with surface area, is less than the sum of the corresponding quantities for the separate droplets, the combination configuration is energetically favored. 
     As shown in FIG. 6, droplets  42  transported by fiber  40  in axial direction  36  may be of a different size or volume from droplets  46  transported by fiber  44  prior to their combination with droplets  44  to produce combined droplets  49 . In fact, within the scope of the invention, there is no restriction on the sizes of particular droplets. Since the chemical composition of the various droplets is controlled, a computer may track them all and control the chemical reactions taking place among the droplets. 
     Probes of Droplet Position and Physical Characteristics Capacitive 
     Variability in droplet position with reference to laboratory-fixed instrumentation may arise because of lengthening or shortening of the fiber due, in turn, to thermal expansion or tensile stress, for example. In order to track the droplet position with great precision, several methods may be practiced. Referring to FIG. 7, a capacitance sensor, designated generally by numeral  70 , is used to measure the capacitance between plates  72 . A capacitance meter  74 , such as known in the art, is connected across the plates and supplies an output signal characterizing the capacitance. The clearance between plates  72  must be sufficient to provide clearance for fiber  12  and droplet  10 , while the width of plates  72  must be no longer than to allow clear resolution of individual droplets. Depending on the dielectric properties of a given droplet  10 , the capacitance measured across plates  72  increases or decreases as droplet  10  enters the region between the plates, as fiber  12  advances in direction  36 . The plot  80  in FIG. 8 depicts the capacitance, plotted along the vertical axis, as a function of the displacement x of fiber  12 . The width w of the capacitance feature corresponds, substantially, to the width of the droplet convolved with the width of plates  72 , and may be dominated by the width of the plates if it significantly exceeds the width of the droplet. Using this technique, the mid-droplet position  82  is accurately determined. 
     Plot  80  of capacitance as a function of fiber position also allows other parameters to be determined. For example, the integral of curve  80  is proportional to the volume of the droplet for a given plate geometry and dielectric constant of the liquid. By making the plate length shorter than the droplet length, the width of the curve yields the droplet length directly, and, otherwise, the droplet width may be derived by deconvolution of the known plate width. Similarly, the dielectric constant of the liquid and the known dielectric properties and geometry of the fiber allow the volume to be derived given an absolute measurement of incremental change in capacitance as the droplet passes through the plates. 
     Similarly, once the droplet volume and length are known, the diameter is readily calculated. 
     Alternatively, if the volume of the droplet is derived by independent means such as by one of the optical methods described below, the capacitance technique described may be used to infer the dielectric constant of the droplet and thus aspects of its material/chemical characteristics. 
     Optical 
     Referring now to FIG. 9, other methods for interrogating the physical and chemical properties of droplet  10  conveyed in direction  36  by fiber  12  employ a microscope designated generally by numeral  90 . The optical arrangement may be confocal (of Type I or Type II) with the interrogated droplet  10  and an aperture stop  92  at conjugate foci of the system, or otherwise, using any optical arrangement known to persons skilled in the optical arts. Using any such arrangement, a source of illumination  94 , which may be a broadband source of light or a monochromatic source such as a laser, provides a light beam  96  that is collimated by collimating optics  98 . In accordance with a preferred embodiment of the invention, beam  96  is focused by focussing optics  100  to form a focus in the vicinity of droplet  10 . A fraction of the light reflected from droplet  10  is deflected by beam-splitter  102  and focussed onto photodetector  104 . 
     As droplet  10  traverses beam  96 , the intensity of reflected light measured at photodetector  104  varies, with optimal performance achieved when aperture stop  92  is matched to the size of droplet  10 . As discussed above, an optical technique may be employed to derived droplet size, whereupon the capacitive technique yields information relating to the dielectric properties of the individual droplets. 
     In accordance with further embodiments of the invention, the light returned from the droplet, whether by reflection or scattering, may be spectrally analyzed, using any or all spectroscopic techniques known in the spectroscopic arts, to derive information regarding the composition and physical state of the liquid comprising droplet  10 . These techniques include fluorescence detection systems based on the fluorescent response, for example, to target drug/protein interactions. 
     Additionally, microscope arrangement  90  may be employed to create an image of droplet  10  at the focal plane where aperture stop  92  is shown. Placing a camera array, such as a CCD array, in the focal plane allows an image signal to be derived, according to standard optical techniques. 
     Longitudinal Propulsion of Droplets Along the Fiber 
     In addition to conveyance of the droplets by moving the underlying fiber, one or more droplets may be moved relative to the fiber. In accordance with one technique for moving a droplet, optical tweezers are employed, using the known effect of dipole forces created by the scattering of light as it passes through a diffractive interface between two dielectric media such as the ambient air and the droplet. The light beam is applied by focussing a laser beam to a confocal zone of the same dimension as the droplet such that the droplet is constrained to remain within the waist of the beam. 
     In accordance with an alternate embodiment of the invention, acoustic pulses are launched from opposite ends of the droplet-laden fiber by means of acoustic transducers such as piezo actuators. The two pulses are designed such that they collide to create an asymmetric longitudinal pulse tending to urge the fiber and thus the droplet preferentially in one direction along the axial length of the fiber. This forms a type of “inertial stepping” as is known in the art of tunneling microscopy for moving solid objects. Using this technique, particular droplets along the fiber may be addressed. 
     Various embodiments of the invention, as described above, may be advantageously employed for sampling droplets from a 96-well plate such as is used extensively in areas of biology, pharmacology, etc. A single fiber may be used for sequentially sampling droplets from each of the 96 wells on the plate. In accordance with an alternative embodiment of the invention, a sampling assembly, such as depicted in FIG. 2A, provides for simultaneous sampling of a plurality of wells by means of a plurality of loops, either of the same fiber or of various fibers. This provides for rapid sampling and conveyance of large numbers of microdroplets, of as many as 96,000 droplets sampled from 1000 plates in the course of on the order of 1.5 hours. 
     Fiber Storage and Analysis 
     Referring now to FIG. 10, a retaining drum  100  is shown which may serve one or more of the following three functions: a) retaining the droplet-laden fiber  12  in such a manner as not to perturb the integrity of the droplets; b) pulling fiber  12  for conveyance of droplets  10  suspended therefrom; and c) providing a platform for analysis, in massive parallelism if required, of suspended droplets  10 . 
     Drum  100  is rotated about its axis  102  by means of a motor (not shown) driving shaft  104 , via any appropriate mechanical coupling. Drum retains fiber  12  in spiral groove  106  which has a pitch equal to the fiber turn-to-turn spacing when the fiber is wound on the drum. Fiber  12  may be taken up or unwound from either end, as shown. In a preferred embodiment, fiber  12  is continuously taken up and paid out, possibly to a further drum for subsequent analysis or to a spool for disposal. A typical pitch may be 500 μm. For an interdroplet spacing of 500 μm, a drum of 100-mm circumference holds 200 samples per revolution, and, with a 500μm pitch, 10 5  samples per 250-mm length of drum may be retained for storage or analysis. Groove  106  has indentations, holes, or depressions (wells) at the droplet spacing so that the droplets are not perturbed by winding of fiber  12  on drum  100 . These holes may be employed for optical transmission between the interior and exterior of the drum to facilitate optical analysis by means of a detector addressing individual droplets, serially or in parallel by means of a detector array. In a preferred embodiment, a stepping motor is employed for rotating the drum, the motor having 100,000 steps per revolution for accurate registration of the drum position. Such a motor provides 1-μm positioning capability with respect to the fiber wound on the drum. 
     It is to be understood that samples  10  may either be retained in adhesion to fiber  12  while wound on drum  100 , or, alternatively, samples  10  may be deposited, in whole or in portion, on drum  100  using fiber  12  for dispensing the droplets onto the drum, whereupon fiber  12  may be removed. 
     Ribbon Transport 
     Referring now to FIGS. 11A-C, an alternate embodiment of the invention employs a flexible transport member for transportation of droplets where the flexible transport member is a tape or ribbon  110 , as shown in cross-section in FIG.  11 A. Tape  110  may be glass, quartz, metal, or various other materials. Amorphous metal (metallic glass) ribbon is preferred since it can sustain tight radii of bending and additionally has a very high tensile strength. These properties are advantage for high-rate sampling, using a loop feed mechanism having pulleys A 1 , B 1 , A 2 , and B 2  as shown. As described with reference to FIG. 2A, flexible member  110  is brought into contact with surface  112  of liquid  114  and droplets adhere to the flexible member. In certain embodiments of the invention, it is desirable to impart a curvature to ribbon  110 . This is achieved by providing respectively convex and concave curved circumferential surfaces  116  and  118  shown in FIGS. 11B and 11C, where pulleys A and B are shown in cross-section. The concave surface  118  may also serve as a recess for straddling liquid droplets borne by the flexible member. 
     Alternatively, as shown in FIG. 12, ribbon  110  may be fully curled in its entirety or in segments, such as to have flat segments  120  and fully curled segments  122 . Full curling forms a cylinder which may be employed advantageously to encapsulate liquid droplet  10 , thereby minimizing or eliminating evaporation of material from droplet  10  during any latency period required in processing the droplet. Ribbon  110  may subsequently be uncurled to provide ready access to liquid droplet  10 . 
     The described embodiments of the invention are intended to be merely exemplary and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in the appended claims.