FR2748567A1 - Positioning biological reagents requiring temperature regulation - Google Patents

Abstract

A pad (120) has transfer elements (122) located on it's surface (124) in a pattern which corresponds exactly to emplacements in the surface of a substrata. The surface (124) is plane and the substrata may be made from metals, semiconductors, paper, glass or plastic. The transfer elements or reservoirs may heated electrically to maintain their temperature In one mode of operation a common biological reagent is transferred from all the transfer elements (122) to each of the substrata emplacements by direct contact obtained by placing the pad (120) on to the substrata surface. Alternatively the pad may take the form of a roller and may incorporate pistons which produce selective transfer.

Description

Headline
METHODS AND SYSTEMS FOR POSITIONING REAGENTS
BIOLOGICALS USING TEMPERATURE REGULATION
Field of the invention
The present invention relates to methods and systems for positioning biological reagents on a substrate.

Technological background
Recently, an increased effort has been made for the development of pellets for the detection of molecules. In general, a molecular sensing patch includes a substrate on which is arranged an arrangement of binding sites. Each binding site (or hybridization site) has its own molecular receptor which binds or hybridizes with a molecule having a predetermined structure. A sample solution is applied to the molecular sensing pad, and the molecules in the sample bind or hybridize with one or more of the binding sites. The particular binding sites at which hybridization takes place are detected, and one or more molecular structures present in the sample are subsequently identified.

 Molecular detection tablets are of great interest for gene sequencing. These pellets, often called DNA pellets, use an arrangement of selective binding sites each having their own single stranded DNA probe. A sample of single stranded DNA fragments, called target DNA, is placed on the DNA pellet. The DNA fragments attach to one or more of the DNA probes according to a hybridization process. By detecting the probes to which a DNA fragment has hybridized, it is possible to determine a nucleotide base sequence in the DNA fragment.

 Different approaches have been developed to position an arrangement of molecular receptors on a substrate. Af fymax proposed a lithographic technique consisting in synthesizing peptides or nucleic acids on a glass surface. To synthesize an arrangement of n-mer oligonucleotide probes, 4n lithographic writing steps are necessary. The reason is that four different constituent nucleotides (adenine, cytosine, guanine and thymine) can be located at each of the n sites of an n-mother probe. A disadvantage of the lithographic technique is that it is necessary to produce a new set of lithographic masks if one wishes to create a new configuration of probes in the arrangement. Furthermore, the use of 4n masking levels undesirably reduces the yield.

 Currently, a molecular sample is applied to probes present on a molecular detection tablet by immersing the tablet in a sample solution. This approach requires a relatively large quantity of samples, samples of which the quantity is often limited. In addition, the molecules will often erroneously bind to sites on the molecular sensing pad and thus produce false positive results.

Brief description of the drawings
Other features of the invention will become more evident and the invention will be better understood by referring to the following detailed description, taken in conjunction with the accompanying drawings, among which
Figure 1 is a block diagram of an embodiment of an apparatus for positioning at least one biological reagent in a plurality of locations on a substrate
FIG. 2 illustrates an embodiment of a member forming a tampon according to the present invention
FIG. 3 illustrates the transfer of at least one biological reagent from the buffer member of FIG. 2 onto a substrate
FIG. 4 illustrates a rectangular tank intended to be used as a transfer element;
Figure 5 illustrates a spherical tank for use as a transfer element
Figure 6 illustrates a way to maintain a temperature in a tank
FIG. 7 illustrates another technique for introducing said at least one reagent into the reservoirs
FIG. 8 illustrates an embodiment of a member forming a buffer which provides another technique for introducing said at least one reagent into the reservoirs
Figure 9 illustrates a tampon member having a plurality of projected portions which define a plurality of transfer members
Figure 10 illustrates an embodiment of the tampon member having a support member which supports an absorbent layer
Figure 11 illustrates another possible approach to increasing the absorbance of the tampon member by increasing an area of a stamping surface.
Figure 12 illustrates an embodiment of the tampon member which uses both projected parts and reservoirs
FIG. 13 illustrates an embodiment of a member forming a tampon having transfer elements which can be selectively projected.
Figure 14 illustrates the tampon member having a non-projected piston and a projected piston
FIG. 15 illustrates an embodiment of a selectively projectable tampon member comprising means for regulating the temperature;
FIG. 16 illustrates an example of a member forming a tampon of cylindrical shape
Figure 17 illustrates a roller used to apply reagent to a tampon member
FIG. 18 illustrates at least one dispensing head used to apply a reagent to a member forming a tampon; and
FIG. 19 is a flowchart summarizing the steps carried out in an embodiment of a method for positioning at least one biological reagent in a plurality of locations on a substrate.

Detailed description of a preferred embodiment
Figure 1 is a block diagram of an embodiment of an apparatus for positioning at least one biological reagent 100 in a plurality of locations 102 on a substrate 104. The apparatus comprises a buffer member 106 on which is applied said at least one biological reagent 100. The buffer member 106 defines a plurality of transfer elements 108 arranged to correspond to the plurality of locations 102. Said at least one biological reagent 100 is transferred from the plurality of transfer elements transfer 108 to the plurality of locations 102 by bringing the tampon member 106 into contact with the substrate 104.

 Said at least one biological reagent 100 may contain a common biological reagent which is applied to all the elements of the plurality of transfer elements 108. In this case, the common biological reagent is transferred to all the locations of the plurality of locations 102 on the substrate 104, and consequently, is common to all of the plurality of locations 102. According to another possibility, each of the transfer elements of the plurality of transfer elements 108 can receive a respective reagent from said at least a biological reagent 100. Here, different biological reagents can be transferred to different locations on the substrate 104.

 It should be noted that said at least one biological reagent 100, in this embodiment as well as in other embodiments described here, can generally comprise any reagent or chemical product. However, biological reagents including, but not limited to, mononucleotides, nucleotide chains such as DNA and RNA fragments, and other nucleic acids are of particular interest.

 Embodiments of the present invention can advantageously be used for the transfer of biological reagents onto a substrate which forms a molecular detection apparatus, such as a pellet for gene sequencing or a pellet for medical diagnosis. Biological reagents can be transferred to form molecular detection probes in binding sites of the molecular detection apparatus. Alternatively, the biological reagents may include target molecules which hybridize to selected sites of the binding sites already having molecular detection probes.

 It should also be noted that the substrate 104 may be made of a variety of materials which include, but are not limited to, metals, semiconductors, paper, glass and plastic, depending on the different embodiments of the present invention described here. The substrate 104 can be flexible or rigid, and can take any shape, including, but not limited to, a planar shape, a roll shape, and a cylindrical shape.

 FIG. 2 illustrates an embodiment of a member forming a tampon 120 according to the present invention. The tampon member 120 defines a plurality of transfer members 122. The plurality of transfer members 122 may be arranged in an arrangement, such as the two-dimensional arrangement illustrated in Figure 2, to correspond to an arrangement of locations on a substrate. It should be noted, however, that other possible configurations of the plurality of transfer elements 122 may be used.

The tampon member 120 has a face 124 on which the plurality of transfer elements 122 is defined. In this embodiment, the face 124 is substantially planar.

 FIG. 3 illustrates the transfer of at least one biological reagent from the buffer member 120 of FIG. 2 onto a substrate 130. The face 124 of the buffer member 120 is directed towards a surface 132 of the substrate 130 until contact is established. When brought into contact, the face 124 is in contact with the substrate 130, or at least with one of the elements of the plurality of transfer elements 122 is in contact with the substrate 130. Then, as indicated by the Reference number 134, the buffer member 120 is distant from the substrate 130. Here, the at least one biological reagent or one of its reaction products remains in a plurality of locations 138 of the substrate 130.

 In the various embodiments of the present invention, the plurality of transfer elements can take any of several forms. In one of the forms, the plurality of transfer elements includes a plurality of reservoirs defined on a surface of the tampon member. The plurality of reservoirs is arranged to correspond to the plurality of locations on the substrate.

Each reservoir has the form of a small inclusion in the member forming a buffer to contain small amounts of the reagents for chemical reactions. For example, reservoirs can be used to contain on the order of a picoliter of a reagent.

 Using conventional alignment techniques to position the mask and patch, at least one reagent can be precisely placed in predetermined locations on a chemically sensitive substrate by contacting the substrate with a buffer member having a corresponding arrangement of tanks. When the buffer member is in contact with the substrate, any chemical reaction between said at least one reagent and the chemically sensitive substrate is confined in the reservoirs. Preferably, the reservoirs have a predetermined shape and a predetermined volume so as to not significantly disturb the chemical reaction of interest.

 The tampon member is kept in contact with the substrate until the chemical reactions are complete. Then, the buffer member is moved away from the substrate, leaving regions of reaction products on the substrate.

 FIG. 4 illustrates a reservoir 140 of rectangular shape intended to be used as a transfer element. The reservoir 140 of rectangular shape is defined in a part of a member forming a buffer 142. In one of the embodiments, the reservoir 140 of rectangular shape has a dimension 144 greater than 10 μm so as not to disturb a reaction predetermined chemical that takes place there.

 FIG. 5 illustrates a tank 150 of spherical shape intended to be used as a transfer element.

The reservoir 150 of spherical shape is defined in a part of a member forming a buffer 152. As for the reservoir 140 of rectangular shape, the reservoir of spherical shape 150 may have a diameter 154 greater than 10 Hm so as not to disrupt a predetermined chemical reaction that takes place there.

 FIG. 6 illustrates a means for regulating a temperature in a tank 160. A heating element 162 is included in a buffer member 164 near the tank 160. The heating element 162 can be a resistive heating element, for example, which heats the reservoir 160 in proportion to a current applied to it.

 A temperature probe 166 is included in the buffer member 164 near the tank 160. The temperature probe 166 measures the temperature of the tank 160 or of a reagent contained in the tank 160. A signal representative of the temperature is communicated to a regulator 168 by the temperature probe 166. The regulator 168 processes the signal to produce a regulating signal which is applied to the heating element 162. The regulating signal is produced in order to maintain the temperature at a desired level.

 By including a heating element and a temperature probe in each tank of a plurality of tanks, it becomes possible to locally regulate the temperature in each tank. This is useful for controlling hybridization of genetic material at specific locations on the substrate for diagnostic purposes.

 Controlling the temperature in a tank is also useful in providing a means of amplifying a biological sample contained therein. In this case, the temperature can be regulated to provide the heating steps in a PCR amplification technique (polymerase chain reaction).

 In the various embodiments of the present invention which are described here, said at least one biological reagent can be introduced into the reservoirs according to many different techniques. One technique is to obtain a substantially uniform wetting of the tampon member. In this case, the buffer member can be immersed in a fluid containing said at least one reagent in order to fill each of the reservoirs. According to another possibility, the face of the tampon member can be rolled on or brought into contact with a saturated absorbent material containing said at least one reagent. In both cases, the same says at least one reagent is typically introduced into all of the reservoirs.

 FIG. 7 illustrates another technique for introducing said at least one reagent into the reservoirs.

In this case, said at least one reagent is placed directly in a plurality of reservoirs of a buffer member 170 using a plurality of corresponding capillaries 172 (or a plurality of corresponding micropipettes). As illustrated, said at least one reagent can be applied to the reservoirs from the bottom of the buffer member 170.

 FIG. 8 illustrates an embodiment of a member forming a buffer 180 which provides another technique for introducing said at least one reagent into the reservoirs. A plurality of capillaries 182 or other similar tubular members are included in the tampon member 180. A capillary 184 of the plurality of capillaries 182 is illustrated in more detail. The capillary 184 is included in the tampon member 180 near a reservoir 186. The capillary 184 is in fluid communication with the reservoir 186. Using this embodiment, a different biological reagent can be introduced into each reservoir of the plurality of reservoirs by a respective capillary among the plurality of capillaries 182.

 The direct positioning methods illustrated in FIGS. 7 and 8 are advantageous compared to the uniform wetting methods both from the point of view of the quantity and of the positioning of the reagents. Specifically, a reduced amount of reagent is sufficient to fill the reservoirs when using direct positioning. In addition, different reagents can be introduced into different reservoirs to be positioned at different locations on the substrate.

 As described above, the plurality of transfer elements according to the present invention can take any of a variety of forms. a second form of transfer elements is illustrated in FIG. 9.

 Figure 9 illustrates a tampon member 190 having a plurality of projected portions 192 which define a plurality of transfer members. The plurality of projected parts 192 is arranged to correspond to a plurality of locations on a substrate on which it is desired to position one or more reagents.

 The plurality of projected parts 192 absorb at least one biological reagent 194 which is applied to one face 196 of the tampon member 190. As illustrated, said at least one biological reagent 194 can also be absorbed by the non-projected parts 198 of the tampon member 190. This can happen if said at least one biological reagent 194 is applied using a uniform wetting technique such as that described above.

 The tampon member 190 is brought into contact with a substrate 200 in order to transfer said at least one biological reagent 194 to a plurality of locations 202. Upon contact, a stamping surface 204 of each part of the plurality of projected parts 192 comes into contact with the substrate 200 at one of the respective locations of the plurality of locations 202. However, the non-projected parts 198 do not come into contact with the substrate 200.

When the buffer member 190 is distant from the substrate 200, said at least one biological reagent 194 remains in the plurality of locations 202 on the substrate 200.

 The tampon member 190 may take the form of a rigid or semi-rigid formed plate made from materials which include, but are not limited to, glass, elastomer, metals, sol-gels, plastic , and polymers. One or more of these materials can be combined in the tampon member 190 for the purpose of achieving flexibility, stiffness, opacity, thermal conductivity and / or desired chemical absorbency.

 FIG. 10 illustrates an embodiment of the tampon member 190 comprising a support member 210 which supports an absorbent layer 212. The support member 210 is formed from a rigid or semi-rigid material, such as those mentioned above. The absorbent layer 212 consists of an absorbent material such as a sol-gel. The absorbent layer 212 defines a plurality of projected portions 214 arranged to correspond to a plurality of locations on a substrate.

 Figure 11 illustrates another possible method for increasing the absorbance of the tampon member 190 by increasing an area of a stamping surface. The tampon member 190 defines a plurality of projected parts 220. Each part of the plurality of projected parts 220 has a stamping surface 222 which defines at least one cavity 224. Said at least one cavity 224 increases the absorbance of each part of the plurality of projected parts 220 by increasing an area of the stamping surface 222. Said at least one cavity 224 can be defined by a series of ridges on each part of the plurality of projected parts 220.

 Figure 12 illustrates an embodiment of the tampon member 190 which uses both projected parts and reservoirs. Here, each part of a plurality of projected parts 230 comprises a stamping surface 232 which defines a reservoir 234. Each reservoir 234 is formed according to any one of the embodiments of the reservoir described here.

 Consequently, each reservoir 234 can contain a precisely controlled amount of the reagent intended to be transferred to a substrate. Furthermore, a capillary can be included in the tampon member 190 for the purpose of introducing a biological reagent into the reservoir 234 of at least part of the plurality of projected parts 230. In addition, a heating element and a probe temperature can be included in the buffer member 190 near the reservoir 234 to provide a means for regulating the temperature for at least one of the projected portions 230. The means for regulating the temperature can be used to provide a means for amplification of a biological sample in the reservoir 234 of at least part of the plurality of projected parts 230.

 The projected parts, in the embodiments of the buffer members described above, can be fixed or selectively projected. Different means for selectively projecting at least a portion of the plurality of projected portions can be used to allow reconfiguration of a single buffer member for a different configuration of locations on a substrate.

 Figure 13 illustrates an embodiment of a tampon member 240 having transfer members which can be selectively sprayed. A plurality of pistons 242 or other similar translation mechanisms are included in the buffer member 240. Each piston of the plurality of pistons 242 can be projected independently so as to form a respective projected portion. Preferably, each piston of the plurality of pistons 242 can be projected electromechanically. Accordingly, an arrangement of transfer elements for the tampon member 240 is electronically programmable based on a plurality of electrical signals.

 The selected pistons of the plurality of pistons 242 are projected to form a particular arrangement of transfer elements. An absorbent layer 244 is deformed by the selected pistons which are projected. The absorbent layer 244 is used to receive a biological reagent intended to be transferred to a substrate. This method can be used to define arrangements having dimensions of the order of a few hundred microns, including dimensions up to 100 Hm or even less. The use of transfer elements which can be selectively sprayed is advantageous for reducing the number of buffers required in a multistage process.

 FIG. 14 illustrates the tampon member 240 having an unprojected piston 250 and a projected piston 252. The projected piston 252 deforms an absorbent layer 254 to form a projected portion 256. The projected portion 256 determines the transfer of a biological reagent to a corresponding location on a substrate 258 which is brought into contact with the buffer member 240.

 Figure 15 illustrates an embodiment of a selectively projectable tampon member having means for regulating the temperature. Here, a heating element 262 is mounted on each piston of a plurality of pistons 260. Optionally, a temperature probe (not specifically illustrated) can also be mounted on each piston of the plurality of pistons 260.

The heating element 262 makes it possible to regulate the temperature of each of the projected parts for the purposes described above.

 In various embodiments of the present invention, the tampon member can take various forms including, but not limited to, a planar shape, a cylindrical shape, a spherical shape, or other curved shapes. The cylindrical shape is advantageous in that it is possible to roll the tampon member on the substrate to transfer said at least one biological reagent to the plurality of locations from a plurality of transfer elements.

 Figure 16 illustrates an example of a tampon member 270 having a cylindrical shape. An outer circumferential surface 272 of the tampon member 270 defines a plurality of transfer members arranged to correspond to a plurality of locations on a substrate 274. Rolling of the tampon member 270 on the substrate 274 causes the transfer of an arrangement of a reagent on the substrate.

 Figure 17 illustrates a roller 280 used to apply reagent to a member forming a pad 282. Roller 280 is well suited for applying a single type of reagent to the entire plurality of transfer members of the member forming buffer 282. The use of roller 280 is also advantageous for applying the reagent to part of the tampon member 282 at the same time as another part of the tampon member 282 transfers the reagent to a substrate 284. Although the member pad 282 is illustrated as having a cylindrical shape, it should be noted that a roller can be used to apply a reagent to pad members having a planar shape or another shape.

 FIG. 18 illustrates at least one dispensing head 290 used to apply a reagent to a member forming a buffer 292. Said at least one dispensing head 290 applies a specific quantity of the reagent directly to the plurality of transfer elements on the member forming pad 292. Said at least one dispensing head 290 is well suited for the application of different reagents to different transfer elements of the member forming pad 292. As with roller 280, the use of said at least one dispensing head 290 is advantageous for applying the reagent to a part of the member forming a buffer 292 at the same time as another part of the member forming a buffer 292 transfers the reagent to a substrate 294.

 In a preferred embodiment, said at least one distribution head 290 is included in a plurality of distribution bars such as those described in the co-dependent application entitled "Method and system for synthesis of oligonucleotides using distribution of specific nucleotides ", which is incorporated by reference in the description of the present invention.

Each of the plurality of dispensing bars is reserved for dispensing a respective type of reagent.

For example, each bar of a set of four distribution bars can be reserved for the application of one of the four respective types of nucleotides. Each of the plurality of dispenser bars has a plurality of individually controlled dispensing heads for dispensing reagent into any of the transfer members of a row of transfer members on the tampon member 292.

 FIG. 19 is a flowchart summarizing the steps carried out in an embodiment of a method consisting in positioning at least one biological reagent in a plurality of locations on a substrate. As indicated in box 300, the method includes a step of providing a tampon member which defines a plurality of transfer members arranged to correspond to the plurality of locations. Preferably, the provided tampon member corresponds to those described herein, although other possible embodiments of the method are not excluded.

 If the plurality of transfer elements takes the form of a plurality of parts projected onto the tampon member, an optional step of selective projection can be performed, as indicated in box 302. Said at least part of the plurality of projected parts can be electromechanically projected by a respective piston in the buffer member, as described above. However, other means for selectively projecting said at least part of the plurality of projected parts can also be used.

 As indicated in box 304, a step of applying said at least one biological reagent to the plug member is carried out. For a transfer member that includes a reservoir, this step may include introducing a biological reagent into the reservoir using a capillary included in the member forming a tampon.

For a plurality of reservoirs, this step can comprise the introduction of a different biological reagent into each reservoir of the plurality of reservoirs by a respective capillary of a plurality of capillaries included in the member forming a buffer.

 As indicated in box 306, a step of bringing the tampon member into contact with the substrate is carried out to transfer said at least one biological reagent from the plurality of transfer elements to the plurality of locations. As described above, the step of bringing the tampon member into contact with the substrate may include stamping or rolling the tampon member on the substrate.

 Since the present invention uses a buffer member which transfers a reagent by contacting a substrate, they offer a significant improvement since the lithographic design is no longer necessary to define chemically active surfaces on the substrate. This makes it possible to increase the yield of a manufacturing process on several levels by reducing the number of stages involved.

In addition, the stamping process is transferable to large areas and compatible with high-speed manufacturing standards.

 The use of reservoirs as transfer elements reduces the amount of reagent required compared to immersing the entire substrate in a solution. The use of pistons which can be selectively projected to define the transfer elements is advantageous in that the buffer member can be reconfigured in real time.

 Furthermore, the various embodiments of the present invention, as described herein, allow precise control of chemical reactions, including hybridization, including the provision of temperature control on each of the transfer items. This reduces the need to maintain a uniform temperature over the entire substrate.

 In addition, the various embodiments of the present invention allow precise positioning of different samples only in predetermined regions on a substrate, such as a molecular detection pad. Limiting sample deposition can reduce the risk of false positive hybridization (or partial hybridization) compared to applying a sample to all of a plurality of combinatorial sites on a detection pad molecular.

 It will be obvious to those skilled in the art that the invention described can be modified in many ways and can adopt many embodiments other than the preferred mode specifically presented and described above.

Claims (10)

 1. Method for positioning at least one biological reagent in a plurality of locations on a substrate, the method comprising the steps of  providing a tampon member which defines a plurality of transfer members arranged to correspond to the plurality of locations; each transfer member comprising a reservoir (300);  applying said at least one biological reagent to a member forming a buffer (304);  regulating a temperature in the reservoir of at least one of the plurality of transfer elements (308); and  contacting the tampon member with the substrate for the purpose of transferring said at least one biological reagent from the plurality of transfer elements to the plurality of locations (306).  The method of claim 1, wherein the step of bringing the tampon member into contact with the substrate (306) comprises rolling the tampon member over the substrate.  The method of claim 1, wherein the tampon member has a plurality of projected portions which define the plurality of transfer elements.  The method of claim 3, further comprising the step of selectively projecting at least one of the portions of a plurality of projected portions (302).  5. Apparatus for positioning at least one biological reagent (100) in a plurality of locations (102) on a substrate (104), the apparatus comprising  a member forming a tampon (106) to which is applied  said at least one biological reagent (100), the member  pad (106) defining a plurality of transfer elements (108) arranged to correspond to the plurality of locations (102), each transfer element comprising a reservoir (160), the buffer member (106) being intended to being contacted with the substrate (104) to transfer said at least one biological reagent (100) from the plurality of transfer elements to the plurality of locations; and a temperature control unit (168) for controlling a temperature in the reservoir (160) of at least one of the plurality of transfer elements (108)  The apparatus of claim 5, wherein the tampon member (106) has a plurality of projected portions which define the plurality of transfer elements.  The apparatus of claim 6, further comprising means for selectively projecting at least one of the portions of the plurality of projected portions.  8. The apparatus of claim 5, wherein the plurality of transfer members (108) comprises a plurality of reservoirs defined on a surface of the tampon member (106), the plurality of reservoirs being arranged to correspond to the plurality of pitches (102)  The apparatus of claim 8, further comprising at least one heating element (162) in the tampon member (106) near at least one of the plurality of reservoirs (160).  10. Apparatus according to claim 8, wherein the contact is maintained between the buffer member (106) and the substrate (104) until the end of the reaction involving said at least one biological reagent (100).