WO2007042972A2

WO2007042972A2 - Method and apparatus for printing a pattern of different reagent fluids on a porous substrate while applying a pressure drop and porous substrate printed with distinct dots having an enhanced depth of penetration

Info

Publication number: WO2007042972A2
Application number: PCT/IB2006/053641
Authority: WO
Inventors: Anke Pierik; Johan F. Dijksman; Willem-Jan A. De Wijs; Antonius J. J. Wismans
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2005-10-07
Filing date: 2006-10-05
Publication date: 2007-04-19
Also published as: WO2007042972A3

Abstract

The invention relates to a method of printing a pattern of distinct dots (40, 62) of different reagent fluids (23) on a porous substrate (8) while a pressure drop is applied across the thickness of the substrate from the printing side (36) to the opposite side (34). The printed porous substrate (8) is used for a chemical assays. The invention also relates to a porous substrate (8) obtainable by said method, wherein the distinct dots (40, 62) of fluid have an enhanced penetration depth. A printing apparatus for carrying out the method according to the invention is also included in the invention. The invention leads to a reduction in the loss of reagent fluid in the printing process and an enhancement of the sensitivity of the substrate.

Description

Method of printing a pattern of distinct dots of different reagent fluids on a porous substrate using a pressure drop, porous substrate printed with distinct dots having an enhanced depth of penetration, and apparatus for the printing method

The invention relates to a method of printing a pattern of distinct dots of different reagent fluids on a substrate, which printed substrate is used for chemical assays. The invention also relates to a printed substrate obtainable by said method and having improved characteristics of the distinct dots, as well as to an apparatus for carrying out the printing method.

US 4,877,745 discloses a system for a method of printing reagent fluids on a printing medium, or in other words, on a substrate. The printing of reagent fluids is required in the manufacture of chemical assay strips. Selected reagents are printed in a desired configuration on strips of paper. The strips may then be used as disposable diagnostic tools to determine the presence or absence of a variety of chemical components.

To perform a chemical assay with a strip, the strip is exposed to a fluid to be tested, such as blood, serum, or urine. Additional reagents may be used in exposing the strip to the fluid to be tested. The interpretation of the test strips after exposure may be simply visual, e.g. by inspecting for a color change. In testing of biological samples, it is common to label these with fluorescent markers. Depending on the type of reagent fluid, a biological sample may adhere to a specific reagent fluid, thus leaving a detectable marking on the substrate. When it is required that the printed substrate is capable of performing various different tests, the substrate consequently has to be printed with a pattern of distinct dots of different reagent fluids. This type of printed substrate is not a subject of US 4,877,745.

In regard of a substrate printed with different reagent fluids, it is a prerequisite that the different fluids are printed on a substrate in the form of distinct dots, i.e. they each occupy a (predetermined) spot which is surrounded by a non-printed area. In order to use the printed substrate for a chemical assay, it is subjected to a post-treatment after the printing, which comprises the washing of the substrate in order to remove all reagent fluid which did not adhere to the substrate and which would otherwise lead to contamination of the assay, and thus to a decrease of the reliability of the test. When applying the known method of US 4,877,745 to the printing of a porous substrate with a pattern of distinct dots of different reagent fluids, it was observed by the inventors that a considerable amount of reagent fluid ejected from a printing nozzle does not adhere to the substrate, so that there is a loss of fluid during the printing process. The non- adhered fluid is considered to be the fluid that did not penetrate into the pores, but instead stayed on top of the surface area. Most of the non-adhered reagent fluid is probably removed in the washing step during post-treatment. Thus, for a given fluid and a given porous substrate, the amount of adhered fluid has its limitations when the known method is used.

Furthermore, it is in general desirable to have an increase in the amount of reagent fluid that adheres to the substrate for a given size of the dot, so that a dot of a given size gives a stronger detection signal when the substrate is used for a chemical assay.

It is an object of the invention to provide a printing method that achieves the elimination or reduction of the above drawbacks of the known method, and to provide a porous substrate that complies more or less with the above general desire.

To achieve this object, the invention provides a method of printing a pattern of distinct dots of different reagent fluids on a porous substrate having a printing side and an opposite side, wherein each distinct dot is printed by ejection of a reagent fluid on the substrate from a nozzle, while a pressure drop is applied across the thickness of the substrate from the printing side to the opposite side.

The invention solves the problem of loss of reagent fluid which does not adhere to the substrate, because the pressure drop applied to the substrate has the effect of enhancing the power with which the reagent fluid is sucked into the pores, thus ensuring a deeper penetration of the reagent fluid into the pores of the porous substrate, so that a larger amount of ejected fluid actually adheres to the substrate. In addition, less reagent fluid is lost during the washing step in the post-treatment after printing because of the deeper penetration of the fluid into the substrate, i.e. the fluid being more remote from the surface area. The pressure drop applied according to the invention should be construed as the pressure on the opposite side being lower than on the printing side.

In practical regard, the substrate is preferably supported on a support facing a printing head comprising a nozzle from which a reagent fluid is ejected, such as is known from a printing apparatus commonly used in the field. The nozzle is in fluid connection with a reservoir of a reagent fluid. If a different fluid is to be printed, the nozzle is brought into communication with a reservoir of another reagent fluid, in any suitable manner familiar to those skilled in the art. The reagent fluids that are printed on the substrates contain mostly biological reagents. Preferably, the reagent fluid is an aqueous solution of a reagent, in general having a micromolar concentration of 1 to 500 . Advantageously, the formulation is cross-linkable in order to graft the reagent fluid to the porous substrate after the fluid has been printed on the substrate.

The substrate is advantageously positioned on the support by substrate positioning means provided on the support, e.g. recesses or projections on the substrate support. The positioning means allow a precise positioning of the substrate on the support, which is of advantage when large numbers of substrates are printed in an automated manner. The porous substrate has in general a thickness of 100 to 300 micrometers, e.g. 120 micrometers. Preferably, the porous substrate is a membrane, which is the common term in the field for a structure that has good permeability characteristics. When such a porous substrate is used for chemical assays, it is preferably positioned as a diaphragm in a flow- through container having an inlet and an outlet, so that a sample to be tested which is passed through the container necessarily also passes through the porous substrate. Because of the cylindrical dimensioning of the flow-through container (in the form of a small tube, for example), the substrates are advantageously circular in shape, so as to fit in a closing manner as diaphragms between the inner walls of the container. The measure of claim 2 has the advantage that the direct force of the pressure reduction is exerted on the opposite side of the substrate and not on the printing side. As a result, the printing process is not directly interfered with by the application of the pressure drop according to the measure of claim 2. Details of this measure are further dealt with in regard of claim 14 below, which relates to an apparatus for performing the method of the invention.

The measure of claim 3 has the advantage that the porous substrate is especially suitable for use in a flow-through container as well as for printing of reagent fluids.

The measure of claim 4 has the advantage of miniaturizing the chemical assay because of the relatively small sizes of the printed distinct dots, allowing smaller sample volumes to be tested. For instance, the diaphragm type of the substrate has in practice a diameter of approx. 6 mm, contains about 130 distinct dots, each dot covering an area of approx. 200 micrometers in diameter. Because of the relatively small dimensioning, the dots can appropriately be named microdots. Obviously, an increase in the amount of dots - up to, for example, 300-400 or even 1000 - printed on a substrate raises the number of tests that can be performed on one (blood) sample. In practice, some differently positioned microdots may contain the same fluid, thus raising the reliability of the test, i.e. lowering the risk of an error in the test. The measure of claim 5 has the advantage that the cross-linking of the reagent fluid grafts the fluid to the substrate, so that the adhesion of the fluid to the substrate is enhanced, and the loss of fluid in the washing step is reduced or eliminated. This also has advantages in using the substrate for a chemical assay because the cross-linking ensures that reagent fluid will indeed stick to the distinct dot during testing. The cross-linking may be achieved in any way known to those skilled in the art, e.g. by emission of radiation such as UV-light, or by applying thermal energy.

The measure of claim 6 has the advantage that a removal of any excess reagent fluid not adhered to the substrate, e.g. by washing with a suitable liquid, prevents, the detection results from being contaminated by loose reagent fluid when the substrate is used for a chemical assay.

The measure of claim 7 - preferably carried out during or after the removal of any excess reagent fluid - has the advantage that the substrate thus obtained, when used for a chemical assay, will enhance the chances that the sample will adhere specifically to the area where the dots of reagent fluid are present. According to a second aspect, the invention relates to a porous substrate printed with a pattern of distinct dots of different reagent fluids, wherein each dot comprises a reagent fluid penetrated into the pores of the substrate, the penetrated reagent fluid having the form of a column in cross-section of the substrate, and wherein the ratio of the height of said column to the covered surface area on the printing side of the substrate exceeds the corresponding ratio for a capillary penetration action of said fluid on said porous substrate.

In other words, the porous substrate according to the invention has the property that, for a given size of a distinct dot on the surface area, a larger amount of reagent fluid is present in the substrate. The sensitivity of a dot is enhanced thereby, which renders possible a detection of smaller traces of substances from a sample, or stronger signals for the same amount of sample. The fact that the sensitivity is raised per given size of a distinct dot allows, from another point of view, a reduction in the size of a distinct dot while maintaining the same sensitivity. Reducing the size of the dots allows the distance between dots to be reduced, and the substrate as a whole may be further miniaturized, or alternatively, more dots can be accommodated on a substrate of a given size. The height of a column of a penetrated reagent fluid according to the invention is distinct from the height obtainable by known printing methods for printing distinct dots of reagent fluids. In these known printing methods, the penetration of the printed fluid into the substrate is mainly governed by the capillary forces exerted by the pores on the fluid. In the known printing method, the height of a column of penetrated fluid is thus limited by properties such as the mean pore size of the substrate, the viscosity of the fluid absorbed, and the power with which the fluid is ejected from a nozzle.

The measures of claims 9, 10, 11, and 12 relating to preferred embodiments of the porous substrate of the invention are the same measures as correspondingly defined in claims 3, 4, 5 and 7, respectively, relating to the printing method. The same advantages as explained for the claims 3, 4, 5 and 7 also apply to claims 9, 10, 11 and 12.

The measure according to claim 13 has the advantage that the porous substrate allows more than 20 tests to be performed with one substrate, which is appropriate for practical purposes. According to a third aspect, the invention relates to a printing apparatus for printing a number of substrates while a pressure drop is applied across a substrate according to the first aspect of the invention. The printing apparatus comprises: a base provided with suspension means for suspending a moveable printing head with at least one nozzle for the ejection of different reagent fluids, which nozzle is in fluid connection with a reservoir of a reagent fluid, said base further comprising a moveable support for supporting a number of substrates to be printed, which support can be positioned so as to face a nozzle of the printing head, the top side of the support comprising openings which are in fluid connection with a pressure-reducing device, and a control unit for coordinating a sequence of steps of: a) a positioning step of positioning a substrate to be printed opposite the nozzle, by moving the support and/or the printing head, b) a printing step of printing a distinct dot of reagent fluid on the substrate by ejecting reagent fluid from the nozzle, wherein the sequence a)-b) is repeated for said number of substrates as well as for different reagent fluids.

The apparatus achieves the above-mentioned advantages of better penetration of fluids into the substrate and a reduction in the loss of fluid during the printing process. In regard of the size of the openings on the top side of the support which are in fluid connection with a pressure-reducing device, the size of an opening is advantageously such that one opening can be fully covered by a substrate.

Furthermore, the apparatus of claim 14 has the advantage that it comprises a device for applying the pressure drop to the substrate, said device directly affecting the opposite side of the substrate and not the printing side, so that the action of the device does not directly affect the printing side, and consequently the printing process is not directly interfered with. The device is, for example, a vacuum pump, preferably having a controllable vacuum.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

In the drawings: Fig 1 is a schematic 3 -dimensional view of an embodiment of the printing apparatus for the method according to the invention.

Fig. 2 is a detailed cross-sectional view of the printing apparatus of Fig. 1 taken on the Y-axis indicated in Fig. 1.

Fig. 3 is a plan view of a printed porous substrate according to the invention, for use in biological analysis.

Fig. 1 shows a printing apparatus 1 comprising a base 2 on which a bridge 4 is mounted in a fixed position spanning over a table 6, on which porous substrates to be printed 8 are placed in a grid pattern of rows and columns. It is noted that, for the sake of clarity, a smaller number of substrates is shown in Fig. 1 than in normal practice, where one batch may consist of several hundreds of substrates applied on one table. Details of the table 6 are dealt with in Fig. 2 below. A print head 10 is moveably connected to the bridge 4 so that it can move in the direction indicated by arrow Y. The table 6 is moveably connected to the base 2 so that it can move through a guiding slot 12 in the direction indicated by arrow X. In the base 2, a controller 15 is provided for controlling the movements of the table 6 and the print head 10. Furthermore, the controller 15 controls the printing from the nozzles (shown in Fig. 2) of the print head 10. The movements of table 6 and print head 10 are actuated by motors which are not depicted and which are controlled by drive signals sent from the controller 15. X, Y, and Z direction are indicated in the right-hand bottom corner for the sake of clarity, and also as a reference for the other drawings.

Fig. 2 shows the bridge 4 on which the print head 10 is moveably attached, the arrow Y indicating the direction of movement of the print head 10. Connected to the print head is an ejection unit 20 having a chamber 22 for a fluid 23 to be ejected as well as a nozzle 24 from which the fluid is ejected. The drawing shows a portion of the table 6 over a range in which three neighboring porous substrates 8 are resting on the table 6. The table 6 is provided with a grid of circular voids 26 surrounded by the table material. The voids have a diameter smaller than the substrates 8 which are laid on the table 6. The voids 26 (also the voids not drawn) are in fluid connection with a channel 28 which is connected to an apparatus 30 for reducing the pressure in the channel 28, and consequently in each void 26. The fluid connection between the voids 26 and the channel 28 is indicated with broken lines. The channel 28 is provided at the lower side of the table 6, the channel 28 being surrounded by a wall 32. The substrates 8 are laid on the table 6 such that each substrate 8 fully covers a void 26. Thus each substrate 8 rests on the table 6 by the peripheral area of its back side 34. The top side 36 of a substrate 8 will be printed upon. Once a lower pressure has been established in the voids 26, a lower pressure will be present at the back side 34 of the substrate 8 than at the top side 36 of the porous substrate 8.A pressure drop is thus created across the thickness of a substrate 8, i.e. the pressure becomes lower from the printing side 36 to the back side 34. When ejected from the nozzle 24, a drop of fluid 23 will land on the porous substrate 8, where a larger portion of the fluid will penetrate the substrate 8 in the Z- direction because of the applied pressure drop. Thus a dot 40 of fluid 23 is printed that can be regarded as a column of printed fluid having a specific cross-section in the X-Y surface area and a specific height in the Z-direction. The ratio of the height of the column to the cross- section is enhanced by the use of the pressure drop according to the invention.

Fig. 3 is a plan view of one porous substrate 8 printed with dots 62 of different fluids according to the invention. The diameter 64 of one dot 62 is approximately 0.2 mm, and the distance denoted dd between two neighboring dots 62 is approximately 0.4 mm. The diameter of one porous substrate 8 is approximately 6 mm and the total number of dots printed on one substrate 8 is 130.

The preferred embodiments indicated above are not to be construed as limiting the scope of the invention, but are merely presentations of preferred embodiments. Also, any reference signs cited in the appended claims are not to be construed as limiting the scope of the respective claims, the reference signs merely serving to illustrate a feature that appears in the appended drawings.

Claims

CLAIMS:

1. Method of printing a pattern of distinct dots (40, 62) of different reagent fluids (23) on a porous substrate (8) having a printing side (36) and an opposite side (34), wherein a distinct dot is printed by ejection of reagent fluid onto the substrate from a nozzle (24), while a pressure drop is applied across the thickness of the substrate from the printing side to the opposite side.

2. Method according to claim 1, wherein the pressure drop is applied by reducing the pressure at the opposite side (34) of the substrate (8).

3. Method according to claim 1, wherein the porous substrate (8) has a mean pore size of 50 to 2500 nanometers, preferably 100 to 500 nanometers, more preferably 300 to 500 nanometers.

4. Method according to claim 1, wherein a printed distinct dot (40, 62) covers an area (64) with a diameter of 1 to 2000 micrometers, preferably 10 to 500 micrometers, more preferably 100 to 400 micrometers.

5. Method according to claim 1, further comprising a cross-linking step wherein the reagent fluids (23) of the printed distinct dots (40, 62) are cross-linked.

6. Method according to one of the preceding claims, further comprising a removal step wherein any excess reagent fluid (23) not adhering to the substrate (8) is removed after the distinct dots have been printed (40,62).

7. Method according to one of the preceding claims, further comprising a blocking step wherein a blocking agent for blocking pores is applied to that portion of the surface of the substrate (8) that is not covered by the printed distinct dots (40,62).

8. Porous substrate (8) printed with a pattern of distinct dots (40,62) of different reagent fluids, wherein each dot comprises a reagent fluid (23) penetrated into the pores of the substrate, the penetrated reagent fluid having the form of a column (40) in cross-section of the substrate, and wherein the ratio of the height of said column to the covered surface area (64) on the printing side of the substrate exceeds the corresponding ratio for a capillary penetration action of said fluid on said porous substrate.

9. Porous substrate (8) according to claim 8, having a mean pore size of 0.45 micrometer.

10. Porous substrate (8) according to one of the preceding claims 8-9, wherein a printed distinct dot (40, 62) covers an area (64) with a diameter of 300 micrometers or less.

11. Porous substrate (8) according to one of the preceding claims 8-10, wherein the reagent fluids (23) of the printed distinct dots (62) are cross-linked.

12. Porous substrate (8) according to one of the preceding claims 8-11, wherein the substrate comprises a blocking agent for blocking pores on that portion of the surface of its printing side (36) that is not covered by the printed distinct dots (40,62).

13. Porous substrate (8) according to one of the preceding claims 8-12, wherein the substrate comprises more than 20 (?) distinct dots (40, 62).

14. Printing apparatus (1) for use in a method of printing a pattern of distinct dots (62) of different reagent fluids on a number of substrates (8), wherein the printing apparatus (1) comprises a base (2) provided with suspension means (4) for suspending a moveable printing head (10) with at least one nozzle for the ejection of different reagent fluids, which nozzle is in fluid connection with a reservoir of a reagent fluid, - said base (2) further comprising a moveable support (6) for supporting a number of substrates (8) to be printed, which support (6) can be positioned so as to face a nozzle of the printing head (10), the top side of the support (6) comprising openings (26) which are in fluid connection (26,28) with a pressure reducing device (30), a control unit (15) for coordinating a sequence of steps of: a) a positioning step of positioning a substrate (8) to be printed opposite the nozzle by moving the support and/or the printing head, b) a printing step of printing a distinct dot (62) of reagent fluid on the substrate (8) by ejecting reagent fluid from the nozzle, wherein the sequence a)-b) is repeated for said number of substrates (8) as well as for different reagent fluids.