WO1998057747A1

WO1998057747A1 - System and manufacturing of small volume transferer

Info

Publication number: WO1998057747A1
Application number: PCT/US1998/011102
Authority: WO
Inventors: William Birch; Alain Carre; Eric Francois
Original assignee: Corning Incorporated
Priority date: 1997-06-17
Filing date: 1998-06-01
Publication date: 1998-12-23
Also published as: US6303387B1; EP0996504A4; CA2288984A1; EP0996504A1; JP2002508704A; US6051190A; EP0996504B1; DE69825591T2; DE69825591D1; CN1260737A

Abstract

The invention comprises using, providing and making at least one transfer rod (10) of radius (R) having a wettable lower surface (12) upon which a liquid drop (16) to be transferred may form. Rod (10) has a non-wettable side surface (14). Initially after molding rod (10) is entierly non-wettable but it is post treated so at least lower surface (12) is wettable.

Description

SYSTEM AND MANUFACTURING OF SMALL VOLUME TRANSFERER

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for the transfer and dispensing small volumes of liquid, especially appropnate in the contexts of biological or chemical analyses and to a method for making the apparatus

BACKGROUND OF THE INVENTION

During the execution of tests or cultures on biological molecules or cell cultures, plates formed from molded thermoplastic mateπal, e g poKcarbonate or polystyrene, are usually used today Usually, the multi-well plate which is used has dimensions of about 80 x 125 mm, and the wells have a diameter of about 8 mm These dimensions are normalized in industry due to the large vanety of apparatuses, which have been developed for automatic analyses The wells of these plates are often filled with a collection of pipettes, which are displaced manually or by robotized device The samples of the products formed in the wells are collected, for example with the aid of a collection of needles, of stainless steel or the tips of plastic mateπal, which are immersed in the wells

Given that it is desirable to carry out a large number of analyses on a single plate, the use of plates having an increasingly large number of wells per plate is growing An increasingly large number of wells on the same standardized plate gives wells of very small volume, thereby it is then necessary to have tools at one's disposal which enable dispensing small volumes of liquid Many devices exist for dispensing liquids in small doses ranging from volumes of one milli ter to fractions of a milli ter Current developments bearing on multi-well plates include progress relating to micro-well and micro-plate technology, it being possible for example to have up to 10,000 wells per square centimeter (see especially US S N 08/747,425) These wells are separated by a distance of about 100 μm. each well having a depth of 15 to 30 μm and a diameter of 20 to 50 μm In order to perform tests with the aid of these micro-plates, it is necessary to be able to carry out accurate transfers of liquid volumes ranging from a thousandth to a millionth of a cubic millimeter, transfers to and from such micro-wells Classical micro-syπnges are unable to manipulate such small volumes, thereby it is necessary to make liquid handling apparatuses, which are conceived in a radically novel manner

A tool is currently on sale, which compπses a matπx of stainless steel pins arranged so that each pin is aligned on a well from a 96-well plate A drop attaches to an individual pin under the action of the forces of surface tension and can then be transferred The tool has 30 precision grooves cut into the pins, near to their tip, for determining the volume dispensed These pins are advertised as capable of dispensing volumes of 1 mm³ and larger to wells or membrane surfaces

Micro-syringes use a liquid reservoir comprising a capillary tube (or liquid chamber) and a piston for dispensing the liquid by pushing it out through a needle Such a system is not suited to the delivery of liquid volumes in the order of a thousandth to a millionth of a cubic millimeter

It would be desirable to

a method at one s disposal for transferring and dispensing volumes in the order of a thousandth to a millionth of a cubic millimeter into micro-wells of a micro-plate with a good reproducibihty (It is recalled here in order to facilitate reading the present text, that 1 mm³=l μl=10 ⁶1) The invention relates to such a method of transferπng and depositing a drop, notably of biological mateπal or of a reagent onto a surface or into a well or a depression as well as the tool associated with said method

It would also be desirable to have a method for making a tool for transferring and dispensing volumes in the order of a thousandth to a millionth of a cubic millimeter into micro-wells of a micro-plate The invention relates to such a method of making a tool for transfernng_^and depositing a drop, notably of biological mateπal or of a reagent, onto a surface or into a well or a depression as well as the tool resulting from the method

SUMMARY OF THE INVENTION

The present invention uses the tip of a solid fiber or rod for depositing microscopic drops having volumes between a thousandth and a millionth of a cubic millimeter The use of a rod or fiber having controlled wetting and non-wetting properties allows "a simple delivery of precisely controlled liquid volumes from a few cubic millimeters to a millionth of a cubic millimeter and less The Applicant has found that, in the case of a non-wettable rod, which has a wettable tip, the volume of a liquid drop formed by dipping the rod into a liquid reservoir is constant and reproducible The volume of the drop can be controlled by the size of the cross section of the lower surface of the rod The larger this size, the larger the volume of the drop that can be suspended from the tip is A small supplementary control of the volume of liquid deposited on the pin can be obtained by varying the depth, the speed of immersion and/or removal of said pin The diameter of the cross section of the lower surface of the rod (diameter of the rod, in the hypothesis of a cylindrical rod) is preferably less than the capillary length of the liquid or in the order of this length Drops smaller than a cubic millimeter, made with a cross sectional diameter of the lower surface of the rod much smaller than the capillary length of the liquid do not expeπence significant influences due to gravity

The object therefore of the present invention is a liquid transfer tool which enables dehveπng a liquid volume of a few cubic millimeters to less than a cubic millimeter into a well or onto a substrate surface (to said transfer is therefore general h associated the distπbution of the liquid, but this association is not however inescapable The drop taken can be dπed on the tip of the rod for analysis ends see later), said transfer tool characteristically composes

- at least one rod having a wettable tip of pre-determined cross section and at least one non-wettable side, and

- a support structure for said rod

Said rod advantageously has its ettable extremity or tip and its non-wettable sιde(s) Advantageously, this is a rod of constant cross section, notably a rod in the form of a cy linder, the radius of its circular cross section generally being between 2 mm and 1 μm with the result that the diameter of said circular cross section is less than oi equal to the capillary length of the liquid

According to preferred embodiments

- said tool of the present invention has several rods whose arrangement and separation are such that they are aligned on the wells distnbuted on a plate of several wells,

- its rod(s) ιs(are) in metal, ceramic, glass. poKmer or in a composite mateπal,

- the tip of its rod(s) is hydrophihc and the side surface of said rod(s) is hydrophobic or the tip of its rod(s) is oleophilic and the side surface of said rod(s) is oleophobic,

- the tip of its rod(s) is coated with a material, which does not adhere biological matenals

Another object of the present invention is the use of said transfer tool, namely methods of transfer and distπbution of small liquid volumes (of a few cubic millimeters to less than a cubic millimeter) according to which - a transfer tool is at one's disposal which has at least one rod whose lower surface of predetermined cross section is wettable and of which at least one side surface is non-wettable,

- said rod is immersed into a liquid-containing reservoir for a pre-determined peπod of time and at a pre-determined depth,

- said rod is removed from said reservoir so that a drop of liquid is retained on the lower surface of said rod,

- said rod is positioned above a receptor medium, and

- the drop is placed in contact with the surface of said receptor medium, (context of the deposit of the liquid m a receptor medium, or

- a transfer tool is at one's disposal which has at least one rod whose lower surface of pre-determined cross section is wettable and of which at least one side surface is non-wettable,

- said rod is immersed into the well (of a multi-well plate) which contains the liquid for a pre-determined peπod of time and at a pre-determined depth

- said rod is removed from said well so that a liquid drop is retained on the lo er surface of the rod,

- said rod is positioned over a receptor container, and

- the drop is placed in contact with said receptor container, (context of liquid removal from a well of a multi-well plate)

It will alread} have been understood that in the first context, the receptor medium is advantageously a multi-well plate and that therefore, within this context, the removed drop is advantageously placed in contact with an internal surface of a well of said multi-well plate Said internal surface of said well is advantageously wettable

In the second context, within the context of an implementation variant, there is also the internal surface of the well, which is wettable

Generally, m one or the other of said contexts, the method is advantageously implemented

- with a transfer tool having several rods, arranged and separated so that they are aligned on the wells distributed on a multi-well plate,

- under the conditions below

+ the depth of immersion of said rod is at least equal to twice the diameter of the circular cross section of the lower surface of said rod,

+ the period of time of immersion of said rod corresponds to a period which is sufficient to allow the liquid to attain an equilibrium configuration or almost equilibrium at the lower surface (the maximum of liquid is thus taken), + said rod is cyhndπcal and the radius of its circular cross section is between 2 mm and 1 μm,

+ the lower surface of the rod is hydrophihc and the side surface of said rod is hydrophobic or the lower surface of the rod is oleophilic and the side surface of said rod is oleophobic

The tools and method of the invention are particularly efficient in ensuπng the transfer of amounts of liquid whose volume is less than about 2 mm³

Yet another object of the present invention is the method of making a transfer tool for the transfer and distribution of small liquid volumes (of a few cubic millimeters to less than a cubic millimeter),

Other characteπ sties and advantages of the invention shall be better understood upon reading the following descnption of implementation examples made with reference to the annexed drawings in which

BRIEF DESCRIPTION OF THE FIGURES

FIG 1 is a cross-sectional view of a pin according to the invention and a liquid drop, which is attached to it,

FIG 2 is a cross-sectional view of a transfer and dispensing tool of the invention, that may be used in implementing the method of the invention,

FIG 3 is a cross-sectional view of a transfer tool which is in contact with a multi-well plate and which deposits liquid drop into the wells,

FIG 4 represents a seπes of photographs of the same pin, before and after 5 successive distributions of liquid,

FIG 5 is a graph, which indicates the variation of the volume distributed onto a wettable surface as a function of the radius of the rod, in the case of a transfer rod according to the invention,

FIG 6 is a graph indicating the vaπation of the volume distπbuted onto a non-wettable surface as a function of the radius of the rod in the case of a transfer rod according to the invention,

FIG 7 is a perspective view of a mold for use in the manufacture of a transfer tool according to the invention,

FIG 8 A is a cross-sectional view of the mold for use in the manufacture of a transfer tool according to the invention,

FIG 8B is a cross-sectional view of the mold shown in FIG 7 A with the removable cover attached to one surface of the mold. FIG 8C is a cross-sectional view of the mold shown in FIG 7B filled with an intrinsically non-wettable material to form the transfer tool,

FIG 8D is a cross-sectional view of the mold filled with the intrinsically non-wettable mateπal with the cover removed, and

FIG 8E is a cross-sectional view of the resulting transfer tool

DETAILED DESCRIPTION OF THE INVENTION

The method according to the present invention is based on the use of one or more rods The term "rod" means any glass, metal, poKmer, ceramic or composite mateπal having the shape of a pm, rod or fiber Generally, such a rod is cylindrical and has a circular cross section of constant radius R It is, however, m no ay excluded from the context of the invention that said rod has a non-circular cross section (ov l, polygonal, ) and/or a variable cross section according to its height The glass or composite material rods are treated so that they are non-wettable by the liquid transferred For example, when a water-based liquid is being transferred, the outer surface of the rods is treated so that it becomes hydrophobic When an organic solvent or other oil-based liquid is being transferred, the outei surface of the rod is treated so that it becomes oleophobic, unless the material is such that its surface is already oleophobic In this case, no surface treatment of the rod is necessary

FIG 1 shows the tip of a rod 10 according to the invention, in cross section The rod 10 has a wettable tip 12 and non-wettable sides 14 A liquid drop 16 is attached to the wettable tip 12 The wettable tip or lower surface 12 is hydrophihc

The non-wetting characteπstic of the sides of the rod and the wetting characteristic of the tip may be obtained in a variety of

and by employing many different techniques Some of the techniques are the following 1) coating an entire rod and cleavmg the rod, 2) coating the entire rod and polishing or abrading its end, 3) coating the entire rod, cutting or polishing the end, and applying a coating to the exposed tip with the aid of a stamp, or by contacting the exposed tip to a surface coating on a substrate, 4) cutting or polishing the end of a rod. contacting said end with a thin polymer film or other non-permeable film or substrate, and coating the sides of the rod by the required hydrophobic or oleophobic treatment, and 5) simph cutting, polishing, abrading, or coating the tip of a rod that intπnsically possesses the desired or required hydrophobic or oleophobic properties

As a non-limiting example, the non-wetting treatment may consist of a coating of a glass rod The coating can be deposited dipping a clean rod in a solution of perflourodecyltπchlorosilane in an organic solvent One liter of solution is obtained by mixing 2 cm³ of perfluorodecyltπchlorosilane in a mixture of 700 cm³ of dried kerosene and 300 cm³ of dichloromethane The resulting coating has a very low surface energy and is non-wetted by most liquids Another simple process of coating the glass with perfluorosilanes such as perfluorodecyltnchlorosilane consists of exposing the glass to the vapor of the silane Following this treatment, the tip of the rod is cut or separated in order to expose a pnstine, untreated lower surface at its tip This untreated lower surface can be wetted, while the sides of the rod remain coated and thereby, non-wettable When the rod is immersed into a liquid reservoir, only its tip is wettable by the liquid As the rod is pulled out of the reservoir, a semi-spherical cap (drop) of liquid is attached by surface tension, only to the wettable surface that has been exposed, as shown in FIG 1

The volume of the liquid collected at the tip of the rod is reproducible and depends little on the depth of immersion of the rod into the reservoir, provided that the rod is withdrawn from the liquid slowly enough to allow the liquid to flow and to et the tip of the rod The volume of the drop of liquid attached to the wettable tip V, is essentialh a function of the radius of the rod, R As long as the depth of the liquid reservoir is se eral times greater than the diameter of the rod (at least equal o twice said diameter), the A, olume of liquid collected at the rod tip is mdependent of the depth of the liquid reservoir

When the liquid drop is transferred to a receiving mateπal having a solid surface, such as a multi-well plate or a flat surface, and when the drop is in contact with the surface of the receiving material, approximately 50% of the volume of the drop is transferred to the solid surface leaving 50% of volume V, on the tip of the rod

The volume of a given liquid transferred by a pm of given size onto a given uniform flat ettable surface at a given speed, with the pin perpendicular to the surface and coming into contact with the surface is constant in the absence of thermal or mechanical fluctuations The fraction of liquid transferred from a pin having a wettable tip onto a wettable surface is approximately 50% This value may vary up to + 10% if the pin is not in full contact with the surface, if the contact speed is increased, or if the wetting properties of the receiving surface vary slightly

Surfaces that show reproducible volume transfers using these techniques include glasses, ceramics, metals and polymers, with the exception of most si cones and fluoropolymers In the case of surfaces that are not wetted by the liquid, the transfer ratio drops to about 10%. or less It should be noted that transfers of liquid onto non-wettable surfaces can be performed but the liquid volume transfer is significantly less than with transfers onto wettable surfaces

The present invention relates to a device intended for transferring liquid volumes between a thousandth and a millionth of a cubic millimeter, with accurate control FIG. 4 represents a series of photographs of a glass rod with a diameter of 125 μm (radius of 62.5 μm) before and after 5 consecutive transfers 1, 2, 3. 4. 5. A volume of 0.24 . 10^" ³ mm³ of tricresylphosphate was accurately transferred to a solid surface of polyethylene terephthalate each time. Upon considering said Figure 4, it is seen that the method of the invention is perfectly reproducible and that about half of the drop transferred has been deposited each time.

The Table below indicates the approximate radius R of the rod. for a transfer of a given V_m, onto a flat surface or onto a micro-well on a multi-well plate. Two solid surfaces are considered, one moderately or highly wettable, labeled V_m (A), and the other which is not wetted by the liquid, for example formed from a silicone or flouropolymer, labeled V_m (B).

It mav be noted that by varying the rod radius within the range of 1 m-9.3 μm, a transfer of any volume may be made onto a wettable surface for all volumes between 1.0 mm and 0.8 . 10^"δ mm³ (between 1 μl and 0.8 pi). The table is a guide, which enables determining liquid volumes transferred by the device specified according to the invention.

Further. FIG. 5 shows that the relationship between rod radius and volume transferred V_m (A)_, is logarithmically linear. The line is plotted from the results in the Table. Thus, one needs only to consult FIG. 5 in order to determine the appropriate rod radius for a desired volume transfer. For larger volumes above 1.0 mm³, it is necessary that the diameter of the rod be increased.

FIG. 6 represents the relationship between the rod radius and the volume transferred V_m(B) when transferring t a non-wettable surface. As in FIG. 5, the relationship is still linear when plotted on logarithmic scale.

It should be noted that V_m may vary slightly with the surface tension of the liquid and its viscosity. Some calibration of the device may therefore be required for the delivery of accurate volumes of a given liquid.

It should be remembered hereby that the transfer device is not limited to cylindrical rods. The rods may have any cross sectional shape including rectangles or squares. Rods ithout sharp corners, for example of circular or oval cross section, are preferred since they have been found to give the best volume reproducibihty The rods do not however have to have a cross section, which is constant throughout all their height

Further, in order to dispense a large volume of liquid, or in order that a given surface be covered, multiple pins forming a matπx may also be used

With the addition of simply engineered support devices, a matπx comprising multiple and parallel rods may be created for the transferπng matπces of drops into micro-well plate structures Preferably, the matrix compπses several rods arranged and separated so that they are aligned with a collection of wells distπbuted in a multi-well plate FIG 2 represents the cross section of a liquid transfer tool 18 havmg a matπx of rods 10 which project downwards from a support 20 Each rod 10 carπes a drop of liquid 16 FIG 3 represents the transfer tool 18 lowered over a plate 22 hav g wells 24 Each rod 10 is aligned with a corresponding well 24 Preferably, the interior of the wells 24 are wettable and the upper surface 26 of the plate 22 is non-wettable In this way, the drop 16 will be drawn exclusively mto a corresponding well 24 and there will be no spill-over onto the surface of the plate The tool 18 is lowered to the point where each drop 16 is in contact with the interior of each corresponding well 24

50% of the volume of the drop is deposited into the well The same process may also be employed in reverse (not shown) Emptv rods from a transfer tool are lowered into liquid containing wells of a multi-well plate Each rod of the tool is immersed in the liquid of a corresponding well Drops are attached to the wettable surface of each rod tip as the rods are removed from the well The liquid drops are then placed in contact with the surface of a receiving container, for example a second multi-well plate, and approximately 50% of the volume of each drop is deposited

It is possible to obtain similar results to those described above by taking a rod that is non-wettable and appl ing a wettable coating to the tip of the rod and the lower parts of the side walls In this way, rods of the same diameter may be able to carr) different volumes of liquid, simply as a function of the height of application of the wettable coating up the side walls of the rod The coating may be applied b\ dipping the rod a certain distance into a solution containing a molecular species to be deposited for example It is noted hereby that said height of application of said coating is not important In any case, rods of this type and their uses make an integral part of the context of the present invention

It should be noted that the use of a transfer as described above can extend to other applications For example, the volumetπcally quantifiable drop at the tip of the rod can be used in a variety of quantitative analysis procedures The drop can be dried and testing can be performed on the residue left on the wettable tip Examples of known tests that can be performed on this residue include NMR, mass spectrophotometry, micro FTIR, time of flight, and matrix assisted laser absorption Testing residue from the tip of a p or rod is known, but one advantage of using a glass rod as a substrate for the residue, for example, is the lack of background carbon

Further, the transfer tool may be used to deliver known volumes of liquid into a miniaturized diagnostic testing apparatus such as that descπbed in European Patent Application EP-A-381 501 (filed under the No 90 301 061 9)

Additionally, the rod tip may advantageously be coated with a coating, which does not adhere biological molecules, based, for example, on polyethylene oxide or polyacrylamide This type of coating serves to prevent the adsorption of biological mateπals such as peptides proteins, nucleic acids, or cells to the rod tip surface

Another embodiment of the present invention is a product havmg a smgle pin having non-wettable sides and a wettable tip held in a pen-like structural support The pipette structure can be held in a user's hand In the same fashion as with a mechanical pencil, a pin is extended from the support structure by a control lever on the top of the pipette After the pin is extended, a cutting edge makes a fresh cut of the pin tip, leaving a fresh exposed wettable surface In this way, a user can make a single transfer, cut and eject the used tip, and have a fresh tip ready for subsequent transfers Ideally, such a device is employed for transfers of liquid in an amount less than or equal to 2 mm³, but could be extended to larger volumes The length of the pin ideally should be sufficient to reach the bottom of a 96-well plate or centπfuge tubes

A further embodiment of the hand held pipetter is a pen-like support structure having a magazine of pins of vaπous diameters and having wettable tips, which are dispensed and ejected with an index finger-controlled stopper Each volume determination pin is preferabh color coded, ring coded, or size coded based on the volume of liquid which can be transferred

A further embodiment of the hand held pipetter is a pen-like support structure capable of picking up pins from a rack and subsequently ejecting them with the aid of a finger lever after use

The hand pipette system descπbed can extend to a matnx or row of pins (therefore comprising more than one pipette)

With reference to FIGS 8A-8E, one method for making a liquid handling and transfer tool for mini or micro-well plates according to the invention is discussed below

First, the mateπal used to make the tool is mixed In this particular embodiment, the material is a two component sihcone rubber, which is intrinsically non-wettable due to its low surface tension (-20 mN m '), although any type of intπnsically non-wettable material could be used In this particular example, the sihcone rubber used is SYLGARD 184 from BASF which is mixed with a curing agent in the ratio of 10 parts in weight of the curing agent for 100 parts of liquid polymer Other examples of sihcone rubber candidates include SYLGARD 182 from BASF or RTV 630 or 615 from General Electric Co

Next, the mold 30 for making the tool 20 is provided as illustrated in FIGS 7 and 8A- 8D The mold 30 is a plate 32 with a plurality of holes 34 which extend through the plate from one surface 36 to the other opposing surface 38 of the plate 32 The diameter of each hole 34 is equal to the desired diameter of pins and the thickness of the plate 32 is equal to the required height of pins to prevent flooding of the rubber tool when the liquid is applied The placement of the holes 34 in the mold 30 corresponds to the placement of the wells in the mini or micro- well plate In this particular embodiment, the mold 30 is metallic although the mold 30 could be made out of other matenals

Next, one face 38 of the mold 30 is temporanh covered with a reπxnable cover 40 as shown in FIG 8B In this particular embodiment, an adhesive tape is used to cover the holes 34 on one surface 38. although other materials to temporarily cover the holes 34 could be used

Once the holes 34 on one face 38 of the plate 32 are blocked, then the material in this particular example sihcone rubber, is poured into the mold 30 as shown in FIG 8C The mateπal is then allowed to set for a peπod of time at room temperature, typicallv ranging between 15 and ^Λ60 hours In this particular embodiment, the rubber is cured at room temperature, e g at about 20°C to 25°C. overnight and is then post-cured at 100°C for 1 hour After the material is cured, the removable cover 40 is taken off the mold 30 which exposes the top surface of the rods or pins 18 for the tool 20 as shown in FIG 8D The removable cover 40 may also be removed before the rubber is post cured at 100°C for 1 hour

The exposed top surface or tips of rods 18 the tool 20 are then treated chemicalK or physically, in order to develop wetting properties Since the other parts of the tool 20 are still in contact with the mold 30, this treatment does not effect those surfaces

way of example, the top surface of the pins 18 can be exposed to the action of an oxygen plasma followed by a chemical grafting of a polar mateπal like silica to develop wetting properties

Once the top surface of the rods 18 have been treated, the tool 20 is removed from the mold 30 and is ready from use as shown in FIG 8E If manufactured properh . the top surface of the rods f8 should be wettable and the remainder of the tool 20 should be non-wettable

One illustrative and non-limiting example of the process of producing a liquid transfer tool according to the ιn\ ention is descπbed below

In this particular example, a rubber transfer tool for a well plate (75 x 1 10 mm²) which has 384 wells, each well having a diameter of 1 8 mm. was made To make this tool, first a stainless steel plate 1 8 mm thick having 384 holes of 1 8 mm of diameter is needed Next, one face of the metal plate is blocked with an adhesive paper Meanwhile the sihcone rubber used to make the tool 20 in this particular example is prepared from about 70 g of polymeπzable SYLGARD 184 mixture {liquid polymer and curing agent) Once the sihcone mbber is prepared, the silicon rubber is poured into the plate The silicon rubber is left to cure overnight at room temperature In the morning, the adhesive paper is peeled off from the mold and the sihcone is post-cured for 1 hour at 100°C

Next, the pin tops of the rubber tool were treated to make them wettable Before unmolding. the pin top of the rubber tool is exposed for 1 minute to the action of an oxygen plasma (power = 100 W. p O; = 0 2 torr, gas flow rate -= 50 cm³ mm ') After the plasma treatment, the rubber (still in its mold) was dipped into a solution of tetramethoxysilane in an acidified mixture of water and ethanol to obtain the hydrolysis of the silane To obtain 1 liter of silane solution, 100 g of tetramethoxysilane were mixed with 100 g of ethanol and 50 g of water acidified with 0 3 g of HC1 12 N This solution was diluted after 1 hour with 570 g of ethanol After dipping the rubber tool in its mold overnight, the rubber in its mold was rinsed with deionized water and then dπed under air flow This treatment rendered the pin tops of the rubber transfer tool w ettable After the descπbed surface treatments of pin tops, the rubber tool was unmolded and readv for use

Very simple transfer experiments with a water based solution demonstrate that the transfer tool made according to this method works as expected, liquid droplets of equal volume being formed on pin tops, the liquid not wetting the pin walls The process described can be adapted to mold different transfer tools with other sihcone rubber materials

Of course vaπous modifications maybe made by the person skilled in the art to the methods and apparatuses which have just been described only as a non-limiting example, without leaving the context of the invention

Claims

What is claimed is

1 In a biological or chemical assay, a method of transferring a quantity of liquid having a volume of less than about 2 μl compπsing the steps of

(a) providing a transfer tool having at least one rod of predetermined diameter having a bottom surface, which is etting and at least one side surface, which is non- wetting

(b) immersing said rod mto a reservoir contaming a liquid for a predetermined period of time and at a predetermined depth,

(c) removing said rod from said reservoir such that a liquid drop is retained on said bottom surface of said rod.

(d) locating said rod above a receiving medium, and

(e) contacting said drop with a surface of a receiving medium

2 The method of claim 1, wherem said receiving medium is a multiwell plate having a plurality of wells

3 The method of claim 2 wherein said drop is contacted with an interior surface of a well in said multiwell plate

4 The method of claim 3 wherein said interior surface of said well is wetting

5 The method of claim 1, wherem said transfer tool has a plurality of rods arranged and separated so as to align with a distribution of wells of a multiwell plate

6 The method of claim 1, wherem said depth is at least twice said diameter of

7 The method of claim 1, wherem said length of immersion is a peπod of time sufficient to allow said liquid to achieve a meta-stable equihbnum configuration on said bottom surface ÷

8 The method of claim 1, wherem the radius of said rod is in a range between 2mm- lμm

9 The method of claim 1, wherem said bottom surface of said rod is hydrophihc and said side surface is hydrophobic 10 The method of claim 1, wherem said bottom surface of said rod is hydrophihc and said side surface is oleophobic

11 A method of transferring a liquid drop from a well of a multiwell plate compπsing the steps of

(a) providing a transfer tool having at least one rod having a top surface, which is wetting and at least one side surface, which is non-wetting

(b) immersing said rod in said well containing liquid for a predetermined time at a predetermined depth,

(c) removing said rod from said well such that a liquid drop is retained on said bottom surface of said rod,

(d) locating said rod above a receiving container, and

(e) contacting said drop with said receiving container

12 The method of claim 1 1, wherein said transfer tool has a plurality of rods arranged and separated so as to align with a distπbution of wells of a multiwell plate

13 The method of claim 1 1, wherein said depth is at least twice said diameter of

14 The method of claim 11, wherein said length of immersion is a peπod of time sufficient to allow said liquid to achieve a meta-stable equilibrium configuration on said bottom surface

15 The method of claim 1 1, wherein the radius of said rod is in a range between 2mm- 1 μm

16 The method of claim 1 1, wherein said bottom surface of said rod is hydrophihc and said side surface is hydrophobic

17 _. The method of claim 1 1, wherein said bottom surface of said rod is hydrophihc and said side surface is oleophobic

18 The method of claim 1 1, wherein said interior surface of said well is wetting

19 A liquid dispensing apparatus for use in biological or chemical assays comprising at least one rod having a radius ranging from approximately 2mm- lμm and having a wetting tip and at least one non-wetting side, said rod having a diameter less than or equal to the capillary length of said liquid, a support structure holding said rod, and whereby said rod delivers a sub-microhter volume of liquid to a receiving medium

20 The apparatus of claim 19, further compπsing a plurality of rods arranged and separated so as to align with a distπbution of wells of a multiwell plate

21 The apparatus of claim 19, wherem said rod is composed of metal

22 The apparatus of claim 19 wherem said rod is composed of ceramic

23 The apparatus of claim 19. wherein said rod is composed of glass

24 The apparatus of claim 19, wherem said rod is composed of a polymer

25 The apparatus of claim 19. wherem said tip of said rod is oleophilic and said side surface is hydrophobic

26 The apparatus of claim 19. wherein said tip of said rod is h\drophιhc and said side surface is oleophobic

27 The apparatus of claim 19. wherem said tip of said rod is coated with a non- binding coating

28 A method of making a tool for transferring and dispensing small volumes of liquid compπsing the steps of

(a) providing a mold having a pair of opposing surfaces, the mold having one or more ϊfoles extending through the mold from one of the opposing surfaces to the other opposing surface,

(b) coveπng the holes on one of the opposing surfaces with a removable cover,

(c) filling the mold with an intrinsically non-wettable material to form the tool with one rod being formed in each of the holes,

(d) curing the intπnsically non-wettable mateπal in the mold, (e) removing the removable cover from the one surface of the mold to expose a tip of the rod in the hole, and

(f) treating the tip of the rod to develop wetting properties

29 The method according to claim 28 wherein the step of cuπng the intrinsically non-wettable mateπal in the mold compπses cuπng the mateπal at a first temperature and then curing the mateπal at a second temperature

30 The method according to claim 29 herein the first temperature is room temperature and the second temperature is about 100°C

31 The method according to claim 28 wherein the intrinsically non- ettable mateπal is sihcone rubber

32 The method according to claim 28 wherein the tip of the rod is treated chemically to develop wetting properties

33 The method according to claim 28 wherein the tip of the rod is treated physically to develop wetting properties