WO2011051562A1

WO2011051562A1 - Membranes for lateral flow assay

Info

Publication number: WO2011051562A1
Application number: PCT/FI2010/050852
Authority: WO
Inventors: Timo Lappalainen; Tuija Teerinen; Marika Kurkinen; Pia Vento; Tomi Erho
Original assignee: Valtion Teknillinen Tutkimuskeskus
Priority date: 2009-10-27
Filing date: 2010-10-27
Publication date: 2011-05-05
Also published as: FI20096105A0

Abstract

The treatment steps presented in this invention provide means to manufacture paper based LFA systems at a lower cost than current products. In addition, both the low cost and printing as a manufacturing method for LFA support the possibilities of using LFA systems in completely new applications, e.g. mass markets for lifestyle products. The scope of invention is to develop cost effective, mass-manufacturable, disposable systems, which exploit the benefits of paper or fibrous structure; e.g. biodegrability, low cost and ability to transfer liquids.

Description

MEMBRANES FOR LATERAL FLOW ASSAY

Background of the Invention Field of the Invention

The present invention relates to membrane strips for lateral flow assays and to the production of the same. Description of Related Art

Lateral flow assay (LFA) tests are based on a highly sensitive and selective binding reaction between the target molecule (antigen) and the corresponding receptor (e.g.

antibody, enzymes). LFA tests are widely used for diagnostic purposes.

The best known LFA test is the human pregnancy test. Other applications are in the detection of toxic compounds on food, infectious diseases, allergens, chemical contaminants and in the testing of drugs of abuse etc. LFA tests are particularly useful in the area of point-of-care testing (POC), which eliminates the need of laboratory work conducted by trained personnel. The liquid sample such as urine, blood, saliva, water or milk is placed on a test device and the test result can be detected visually in 5-30 minutes.

A conventionally designed lateral flow strip test is shown in Figure 1. It is composed of a reaction membrane 1, a sample pad 2, a conjugate pad 3 and an absorbent pad 6. The parts overlap onto one another and are laminated to the backing material 7 using a pressure- sensitive adhesive. From the sample pad 2, liquid is transferred onto the conjugate pad 3. The primary function of the conjugate pad 3 is to hold the detector particles in a dry state so that they are functionally stable until resuspended by the sample. The absorbent pad 6 is placed at the distal end of the test strip. It absorbs excess liquid from the lateral flow membrane and holds it for the duration of the test. The absorbent pad 6 is often made out of cellulosic paper. Backing 7 improves physical strength of the test strip and also prevents liquid flow out through the bottom of the components. In lateral flow immunoassay tests (LFIA) antibodies to the analyte are used for recognition. Immunoglobulin loaded label particles are utilized to make antigens visible to the eye. The two main formats of the LFIA test are competitive and direct (i.e. sandwich) formats. In a direct LFIA test, an antibody specific for the analyte is immobilised on the membrane at the test line 4. The detector reagent, typically an antibody coupled to coloured latex or colloidal gold particles is deposited and dried on the conjugate pad 3.

In practice, when a test is run, a liquid sample which contains the analyte of interest (e.g. an antigen) is added to the proximal end of the strip, i.e. the sample pad 2. The liquid sample migrates to the next zone, the conjugate pad 3, where it re-mobilizes the dried labelled conjugate. Then, the analyte of interest (e.g. the antigen) interacts with a labelled conjugate. Both migrate into the next section of the strip, which is the reaction matrix. The reaction matrix is conventionally made out of nitrocellulose. Thus, the analyte present in the sample is bound by the antibody coupled to the coloured label particle. As the sample passes the test line, the analyte-detector reagent is trapped and the accumulation of coloured label particles results in the appearance of specific colour lines. The colour intensity of the test line is normally directly proportional to the amount of analyte present in the sample. The control line 5, which is usually deposited after the test line in the direction of flow, confirms correct test development. If the test is performed properly a control line should form, irrespective of the result on the test line.

Currently, membrane strips for lateral flow assays (LFA) are produced from synthetic polymeric materials, such as nitrocellulose, nylon, polyethersulfone and polyethylene, or from fused silica, nitrocelluose (in the following abbreviated NC) being the most widely used material. Nitrocelluloce is in use in very commercially available LFA tests.

The manufacture of NC membranes has been developed for several decades.

Characteristics that make NC membranes useful in LFA applications include relatively low cost, true capillary flow characteristics and high protein binding capacity.

One problem related to NC membranes is the relatively low mechanical strength. This property limits the roll-to-roll processability of the material, although roll-to-roll processes for NC LFA exist. Further, in view of the great consumption of membrane strips in LFAs, there is a need for alternative membranes which are still more inexpensive. Summary of the Invention

It is an aim of the present invention to eliminate at least a part of the problems relating to the art and to provide a novel membrane material for use as membrane strips in LFA applications.

It is another aim of the present invention to provide a method of producing novel membranes for LFA applications. Many patents have been filed in the field of LFA membranes. In the publications, paper is mentioned as one interesting membrane material. So far, paper membranes have not, however, been made commercially availble for this use. One reason is that ordinary, commercial paper grades are not suitable for use in LFA tests. In connection with the present invention it has been found that specific grades of papers, viz. cellulose papers produced from short-fibered pulp, exhibit interesting properties and show great potential as membrane materials. In particular, it appears that papers which are based on bleached, short-fibered pulp have two or more characteristics selected from the group of high formation, high brightness, good wet strength and excellent lateral flow speed of the membrane. These characteristics are important for the use of the cellulose paper as a membrane strip material in LFAs.

Based on this finding, a lateral flow assay test strip can be provided, which comprises in the flow direction a selectively activated membrane for a test reaction and an absorption pad, wherein both the selectively activated membrane and the absorption pad are made from a paper strip material produced from bleached, short-fibered pulp of deciduous tree.

More specifically, the use according to the invention is characterized by what is stated in claim 1.

The lateral flow assay test strip is characterized by what is stated in the characterizing part of claim 20. Considerable advantages are provided by the present invention. Thus, use of bleached cellulose papers, optionally modified to improve wet strength and porosity, as membranes allows for the manufacture of a new type of LFA products, because paper is mechanically much stronger than unbacked membranes made of nitrocellulose and manufacturing of paper in large scale is much more inexpensive than that of nitrocellulose.

Use of printing methods in applying the detection molecules on the substrate gives possibilities to pattern the detection area in a decorative way, and to combine this with printed text and graphics.

Capillary flow characteristics of paper membranes can be adjusted also very easily. Protein binding capacity of fibrous structure can be increased to the sufficient level by

functionalization treatments. In addition to that mechanical properties of dry paper are significantly better than that of nitrocellulose membrane, which improves the rapid roll-to- roll fabrication possibilities of the LFA products.

Use of paper as a membrane gives possibilities to manufacture new type of LFA

products. Whereas traditionally designed assays are composed of a variety of materials, each serving one or more purposes and in which the parts overlap onto one another and are mounted on a backing card using adhesive, the present invention gives possibilities considerably to simplify the structure of LFA tests. One method to simplify LFA test is presented already in the co-pending Finnish Patent Application No. 20095503, the contents of which are herewith incorporated by reference. In our earlier invention part of the bioactive paper strip can be used as a lateral flow membrane and the distal end of the strip is mechanically modified to function as an absorbent pad.

One particularly interesting application lies in the development of immunoassay test to detect contaminants in food and water and small animal segment (bacterial/viral infections, fertility issues). The present technology can also be used to assays employing the lateral flow principle, e.g. in enzyme or nucleic acid based assays, and in assays based on chemical reaction.

It is conceived that in the future manufacturing of printed paper products, where one sheet contains a LFA test, will potentially be possible. Compared to nitrocellulose membranes where flammability and poor mechanical strength is an issue, cellulose papers provide a much more viable solution to the production of such test strips. The present technology provides for potential LFA tests for large- volume applications, such as home testing, rapid point of care testing, and in the field for various environmental and agricultural analytes. Market segments for LFA in which paper based membranes can be used are cases, where need to perform very large number of test/day exists. Those segments include biodefence and environmental testing. In addition, completely new markets in household and lifestyle related industries can be developed based on the present technology. As the example below shows, by selecting a suitable cellulose pulp as a starting point, a cellulose paper, when additionally and optionally treated with chemicals for modifying its properties, and manufactured to a suitable grammage, has properties which are similar to those of a nitrocellulose membrane. In fact, in one embodiment, the properties of a cellose paper which are of importance for its present use (cf. functional properties 2 and 4 - 7 of table 1) are tailored by the selection criteria and treatment step discussed below such that they do not deviate from those of a nitrocellulose membrane by more than about ± 20 %.

Tailoring of the properties of a cellulose paper allows for its use as a replacement of a conventional nitrocellose membrane. The cellulose paper can be used as such or provided with a backing layer.

Thus, in summary, the present invention provides cost effective, mass manufacturable, disposable systems, which exploit the benefits of paper or fibrous structure; e.g.

biodegrability, low cost and ability to transfer liquids.

Description of Preferred Embodiments

Next, the invention will be examined more closely with the aid of the following detailed description. Figure 1 shows, in a schematic, perspective depiction, the main parts of a lateral flow assay strip;

Figure 2 shows, similarly in a schematic, perspective depiction, an embodiment wherein a part of the paper membrane is used as a lateral flow membrane and the distal end of the strip is modified to work as an absorbent pad;

Figure 3 is a digital image of the visual tests for hemoglobin; and

Figure 4 shows a photograph of the hydrophilic channel on 80 g/m² eucalyptus paper.

As discussed above, the present invention provides for the use as membrane strips in laminar flow assay of paper, in particular paper produced from bleached, short-fibered pulp of deciduous trees.

Several paper properties are important with regard to the use of paper as a membrane of LFA test:

(1) Lateral flow properties. Capillary flow rate affects the sensitivity of the membrane, because it determines the length of time period when antigen and antibody are close enough to bind each other. (2) Immobilization of receptors to fibrous network. Membrane must irreversibly bind capture reagents at the test line.

Therefore high and consistent level of protein binding is needed. Functionalisation of fibrous structure is used in order to increase immobilization of proteins to the fibrous network.

(3) Blocking of the fibrous network. Blocking of the membrane is used to prevent nonspesific binding of the detector particles and analyte. Immersion methods are often in use, when blocking membranes. The addition of of blocking agents has to be done on dry web, because blocking agents are normally applied after the addition of antibodies on the test line. Therefore the wet strength of paper membrane must be good.

(4) Optical and surface quality properties. Good and uniform optical and surface quality of fibrous network is needed, in order to achieve a clear test result. The brightness of the test strip is important for the visual evaluation of the immunoassay test. In addition to that customers make demands on optical and surface quality properties of bioactive paper, especially when it is question about pharmaceuticals. The readability of the signal depends on the contrast between the test line and the adjacent membrane area.

It has been found that paper based on short-fibered pulp of deciduous tree is capable of meeting a number of the listed properties. Firstly, formation of the paper is good.

Secondly, this kind of paper has consistent flow characteristics and modification of wicking rate is easy. Thirdly, wet strength of paper membrane is also good, which is important because some of the functionalization treatments and blocking of paper membrane are done to dry paper. Fourthly, the mass-manufacturability using roll-to-roll printing or printing like methods (gravure, flexo, inkjet or screen printing, coating and soaking) of the final LFA assay, a highly valuable property, is also present. Finally, the present paper has a good and uniform optical and surface quality of the fibrous network, which provides for a clear test result. Cellulose papers are commonly used for sample and adsorption pads. The present technology describes how fibrous network can be further modified in order to allow for efficient use of paper as a membrane material.

Thus, the various parameters relating to selection of pulp type and optional modifications of the pulp or the paper will next be discussed.

Selection of appropriate pulp type is an important step in the manufacturing of paper membrane. Pulp type affects formation, brightness, wet strength and lateral flow speed of the membrane. Formation is an extremely important property of the paper membrane. If the paper formation is poor there can be large local variations in several paper properties causing variation between test strips. Therefore the reproducibility of performance can be poor.

In the present novel technology short fibered pulps are used. They give better formation than long fibered pulp. Therefore eucalyptus is a particularly suitable wood raw material for paper membrane. However, acacia, poplar, aspen, alder and birch are equally suitable. In one embodiment, a pulp is selected having a specific formation index (Sfi) of the paper membrane of about 0.2 to 1.2 Vg/m², in particular (for handsheets) about 0.2 to about 0.5 Vg/m², for example about 0.40 Vg/m² ± 20 %. This applies to papers and paper sheets having a grammage of about 35 to 120 g/m². For example, the specific formation index (Sfi) of a paper membrane made of unbeaten, bleached, eucalyptus pulp, in particular one having a grammage of 80 g/m², is 0.40 Vg/m².

In addition to good formation, also the brightness of the paper used as a membrane should be good, in general higher than about 88.5 %, in particular higher than about 90 % (ISO). For example pulp of bleached, unbeaten eucalyptus is about 91 %. This value is only slightly smaller than that of nitrocellulose (94-95 %).

Based on the above, one embodiment comprises selecting a pulp which has been produced by a chemical pulping method, in particular an alkaline pulping method, preferably an alkaline pulping method capable of removing lignin from the pulp. A specific example of a suitable pulping method is the traditional or extended sulphate pulping, in batch or continuously. The pulp is preferably bleached. The bleaching process can be carried out using bleaching chemicals selected from the group of chlorine dioxide, peroxide chemicals and ozone and combinations thereof.

In the following, specific reference is made to eucalyptus pulp. It should still be noted that the description is applicable to pulps of other deciduous tree species.

According to a preferred embodiment, the paper strips have a fibrous matrix which comprises pulp of the above kind. According to another embodiment, the papers which are used as membranes consist of, or consist essentially of, pulp of the above kind. Thus, the membranes may contain in addition to the fibrous material of the pulp also various components which modify the properties of the material so that it is better suited for use in LFA.

Preferably the density of a handsheet made of the unmodified pulp is less than about 600 kg/m³. "Unmodified" stands for a pulp which has not been treated with any modifying chemicals as will be described below.

In an embodiment, the pulp is used in unbeaten state. In another embodiment, the pulp has been gently refined. Preferably the gently refining comprises steps of improving at least one property selected from flexibility, fibrillation and surface properties, while essentially not changing the fibre length of the pulp.

The wet strength of higher grammage paper is higher than that of smaller grammage paper. But on the other, hand lateral flow speed decreaces as the grammage of paper increases. Therefore in one embodiment, the grammage of the paper is about 40 to 120 g/m², preferably about 60 to 100 g/m², for example about 70 to 90 g/m², in particular about 80 g/m², when using bleached, unbeaten, eucalyptus pulp. As mentioned above, the same grammage is applicable to pulp of other deciduous tree species.

Refining of the pulp influences the lateral flow properties of paper samples. However, addition to various additives, such as cellulose derivatives, such as cellulose ethers and similar dispersing agents, is another - optionally additional - method to efficiently modify the lateral flow properties of the paper samples.

In one embodiment, CMC (carboxymethyl cellulose) is added to the pulp. As a dispersing agent CMC affects on the drainage of pulp. Therefore in mass manufacturing of bioactive paper the fine-tuning of lateral flow properties can be done by CMC addition. The combination of refining and different dosages of CMC gives a mean to produce

membranes having different wicking rates.

Air permeability of an unbacked, optionally chemically modified (as described above) paper strip is, according to one embodiment, in the range of about 5300 ml/min ± 20 %. In addition to refining and CMC addition there are also other methods to modify lateral flow properties of paper membrane. In order to increase porosity of paper membrane use of once dried pulp is recommended instead of never dried pulp. Porosity and therefore lateral flow speed of paper membrane can be increased by adding surfactant (e.g. polyvinyl alcohol or polyoxy alcohol) to the paper making pulp. Flow characteristics of paper membrane can be also modified by using additives like starch and starch derivatives, which causes changes to the hydrophilic properties of paper membrane. Furthermore intensity of wet pressing has also very strong effect to the porosity of the paper membrane. Use of impingement drying and also in small scale calendering can be used to modify porosity of paper membrane causing changes to the lateral flow properties of paper membrane. Very porous paper structures can be made by using freeze-drying method. A need to improve wet strength properties of paper samples exists, because some of the functionalization treatments and blocking of the paper membrane are done to dry paper samples. The structural integrity of the paper weakens, due to the rewetting of paper. Wet strength properties of wet paper samples can be improved by using wet strength chemicals. The mechanism by which they provide wet strength is through bonding to or encapsulation of the cellulose fibers to provide a water-resistant, hydro lytically-stable, polymer- reinforced cellulose fiber network. In one embodiment, wet strength chemicals were used. The chemicals can be selected from, e.g. the group of polyamideamineepichlorohydrin (PAE) resin and urea- formaldehyde (UF) resin and mixtures thereof.

In one embodiment it was found that sufficient wet strength can be achieved by using wet strength chemicals of the PAE type in dosages of 1 up to a mximum of about 20 g/kg fibrous matter, in particular about 2 to 5 g/kg fibrous matter, for example about 2.5 g/kg fibrous matter. Alternatively or additionally, UF resin type chemicals can be added in dosages of 1 to 50 g/kg fibrous metier, in particular about 2 to 30 g/kg fibrous matter, in particular about 5 to 20 g/kg fibrous matter, for example about 10 g/kg fibrous matter. A PAE resin (dosage 2.5 g/kg) or UF resin (dosage 10 g/kg), was sufficient when paper is made of eucalyptus pulp. Those dosages of wet strength chemicals do not decrease significantly the lateral flow speed of particles that are present in the flow stream. Larger amounts of wet strength chemicals would seem to decrease lateral flow speed of particles. In an embodiment, the properties of the cellulose paper (in handsheet form) have been modified by a combination of the above explained steps such as to obtain a paper strip having a capillary flow time of water which largely corresponds to that of the substrate now commonly in use (nitrocellulose, 315 s/40 mm for a strip having a grammage of about 40 g/m²). More precisely, in a preferred embodiment, the capillary flow time of water for a present paper or handsheet is - as such - about 300 s/40 mm ± 40 %, in particular, 300 s/40 mm ± 30 %, preferably about 300 s/40 mm ± 25 %.

Cellulose contains functional side groups that can be exploited in functionalization and immobilization. According to one embodiment, chemical agents capable of reacting with the functional groups are used to provide surfaces capable of binding proteins and other analytes.

In a preferred embodiment, carbodiimide (CDI) and periodate are used for activating hydroxyl groups of cellulose.

Immobilization of proteins at cellulose surface can be achieved through the conjugation of activated groups to the amino groups of proteins. Immobilization can be carried out during antibody printing process, which enables high-throughput fabrication.

A blocking step, which in one embodiment comprises the step of soaking the membrane with blocking substances, can be performed to prevent the nonspecific binding of proteins. The blocking step can be carried out by printing or coating methods and therefore it can be included in the LFA roll-to-roll fabrication process.

For the sake of completeness it should be pointed out that the pore size distribution of paper membrane differs from that of nitrocellulose. Due to the smaller pore size large, conjugated latex beads are generally not capable of flowing into the paper membranes. However, according to one embodiment of the invention, colloidal gold particles or other small-sized labels are used with the paper membranes

The treatment steps presented in this notification of invention provide means to

manufacture paper based LFA systems at lower costs than the current products. In addition, both the low cost and printing as a manufacturing method for LFA support the possibilities to use LFA systems in completely new applications, e.g. mass markets for lifestyle products.

Mass production methods of paper give possibilities to decrease manufacturing costs of LFA membranes significantly. Capillary flow characteristics of paper membranes can be adjusted also very easily. Protein binding capacity of fibrous structure can be increased to sufficient level by functionalization treatments. In addition to that mechanical properties of dry paper are significantly better than that of nitrocellulose membrane. Figure 1 has already been discussed above. It shows a typical LFA format and the use of it in analysis.

The following reference numerals are used in the drawings (Figures 1 and 4):

1, 11 Membrane

2, 12 Sample pad,

3 Conjugate pad,

4, 14 Test line

5, 15 Control line and

6, 16 Absorbent pad

7 Backing

In paper-based LFA all compartments (reference numerals 2, 12; 3 and 6, 16 in the drawings) can be fabricated from paper by adjusting physical properties of each. Paper has to be attached to a water-resistant layer 7 in order not to allow liquid flow out of the bottom of the membrane.

Figure 2 illustrates an embodiment, wherein a part of the paper membrane can be used as a lateral flow membrane and the distal end of the strip is mechanically (and chemically) modified to function as an absorbent pad.

As stated earlier, the problem with nitrocellulose is that it is mechanically very weak and its structure cannot be modified to be suitable for use as an absorbent pad. The use of a separate absorbent pad requires very accurate positioning and gluing of the absorbent pad. On the other hand paper membranes are mechanically stronger. The absorbency of a paper membrane can be modified mechanically during the converting process; by embossing and by folding the paper strip several times. When folding the paper several times a layered z- directional paper-air-paper structure having high absorbency is created (see Fig 2). The embossing creates "void areas" allowing the strip to hold more liquid. In other words, the liquid absorption properties of the distal end of the paper test strip are modified in such a way that the distal end of the paper strip works as an absorption pad. In this case there are no overlaps. Therefore the manufacturing of the immunoassay test is easier and the manufacturing costs are reduced. Thus, based on the above, in an embodiment, the absorption pad of the membrane is formed by a portion of the paper strip which is embossed or folded to increase the absorption capacity of the strip. The absorption pad can comprise transversal paper strip folding to provide a plurality of adjacent paper layers.

The following non-limiting example illustrates the use of one particular embodiment of the present invention.

Example

As a raw material of a test strip bleached eucalyptus pulp was selected. The eucalyptus pulp was obtained from a pulp mill and comprised a pulp produced by the sulphate method (Kraft pulps) and bleached using ECF method (elemental chlorine free). This chemical pulp had been delivered as dry pulp sheets. The eucalyptus pulp, with short fibres and a narrow fibre length distribution, had good formation as necessary for the paper membrane. A 80 g/m² eucalyptus paper was selected, because its properties were close to the properties of a typical nitrocellulose membrane. The paper was produced from an unbeaten, once-dried pulp and contained a wet strength improving chemical (PAE) in a dosage of 2.5 g/kg dry matter (fibres). When paper was made of eucalyptus pulp sufficient wet strength was achieved by using a dosage of 2.5 g/kg PAE resin or a dosage of 10 g/kg UF resin. Those dosages of wet strength chemicals did not have a detrimental effect on the lateral flow properties of the paper membranes

Table 1. Comparison of properties of paper strips of unbeaten, bleached Eucalyptus kraft pulp (handsheet of 80 g/m² grammage, wet strength improved with 2.5 g/kg of PAE) and nitrocellulose (39 g/m² grammage)

The use of 80 g/m² paper made of unbeaten, bleached eucalyptus pulp with a dosage of 2.5 g/kg PAE as a membrane material of the LFIA hemoglobin test is demonstrated in the following: Fabrication of a paper-based lateral flow test was demonstrated by developing a noncompetitive immunoassay for hemoglobin. The hemoglobin immunoassay was selected as a model due to its wide use in rapid point-of-care diagnostics (Sanford, McPherson 2009). Figure 3 shows test strips carried out with hemoglobin standards over the concentration range of 0-250 ng/ml. Colloidal gold conjugated to anti-hemoglobin 7202 antibody was used for visual assessment of the test. The amount of capture antibody on the test line was 1 μg on a 5 mm- width test strip which can be considered sufficient to allow the formation of immunocomplex also in the highest concentrations of hemoglobin.

However, it is unknown how much of the capture antibody still remained on the test and control lines after washing with blocking liquids during the test procedure. Sample (Whatman Fu-5) and absorbent (Whatman CF6) pads, commonly used and developed for nitrocellulose membrane, were selected for the paper-based lateral flow test. Whatman Fu- 5 was chosen due to its low protein binding characteristics, which was favourable in the release of antibody-conjugated colloidal gold. Whatman CF-6 was preferred as an absorbent pad owing to its relatively high water absorbing capacity. For simplicity, anti- hemoglobin gold conjugate was applied on the sample pad with the hemoglobin sample instead of first drying the conjugate on a separate pad. An adequate release of the conjugate from the sample pad was observed.

The composition of the blocking buffer had a profound effect on the speed of the test and on the flow of conjugated colloidal gold on the paper membrane. However, by modifying the buffer compositions it was possible to select a buffer which gave a proper flow and assay sensitivity. The optimal blocker consisted of 3% BSA and 0.1% Tween in a 0.1 M PBS. Obviously, these high concentrations of blocking agents were advantageous for the type of paper developed in this work, because without any blocker, the determined flow rate was high compared to the types of nitrocellulose with the highest flow rates.

Figure 3 shows digital images of the visual test for hemoglobin. The LFIA was performed in a one-step sandwich format. The immobilized amounts of anti-hemoglobin (on test line) and anti-mouse (on the control line) were 1.0 μg/5 mm. Different concentrations of hemoglobin standards are indicated.

Thus, it was found that the use of high concentrations of blocking agents compensates the higher flow rate of the eucalyptus paper. The composition of blocking buffer also affected the appearance of the visible signals on the test and control lines. The lower flow rate gave more intense colour on the test line and thus higher sensitivity. The signal generation took 20 min, which is a fairly long time for a rapid test. It was observed that if the paper was not blocked the flow rate of the sample buffer was faster; it took 1 min for, the liquid front to reach the end of the paper strip and the absorbent pad. Whereas with the buffer containing 3% BSA and 0.1% Tween in a 0.1 M PBS the wicking time was 2 min and in even higher concentrations, up to 5% BSA in a 0.1 M PBS, the wicking time was as high as 5 min.

Without any blocking step the sensitivity of the hemoglobin assay and intensity of the test line were somewhat lower. The intensity of the colour development on the test line was proportional to the amount of hemoglobin present in the sample at lower concentrations of hemoglobin, i.e. 0 to 100 ng/ml, and as such the paper-based lateral flow immunoassay can be regarded as a quantitative test for hemoglobin. In immunodiagnostic tests the cut-off level of 100 ng/ml of hemoglobin is often suggested. The lowest detectable signal was observed at 10 ng/ml and therefore, the developed paper-based test can be presumed to meet the required sensitivity of a diagnostic test. Hydrophilic microchannels on 80 g/m² paper membrane, made of unbeaten, bleached eucalyptus pulp with PAE (2.5 g/kg), were generated using the method presented in literature (Martinez, A., Phillips, S.T., Wiley, B., Gupta, M. and Whitesides (2008): H.M. FLASH: A rapid method for prototyping paper-based microfluidic devices. Lab Chip (8), 2146). Hydrophobic regions were made on hydrophilic paper using photolithography and conventional photoresist (SU-8). It was noticed that our 80 g/m² eucalyptus paper sustains washing with acetone well and more importantly the remaining polymerized photopolymer extends >150 μιη through the thickness of the paper. According to the literature cited above, the penetration of polymerized photopolymer has normally been limited to 70 um.

When evaluating the sample visually, it was noticed that the wall profiles of the microfluidic channel was good. Figure 4 shows a photograph of the hydrophilic channel on 80 g/m2 eucalyptus paper. The width of the microfluidic channel is 6 mm. The sample was imaged using a backlight. The transmission of light is higher on the hydrophobic area than on the hydrophilic area.

The capillary flow time of the hydrophilic area (microfluidic channel) was increased by about 70% due to the treatment. No change in the wet strength was noticed. The time to break of the paper was 45 minutes both before and after the treatment.

Claims

Claims:

1. Use of paper produced from bleached, short- fibered pulp of deciduous trees as a membrane strip in laminar flow assay.

2. The use according to claim 1, wherein the paper has a fibrous matrix which consists of, or consists essentially of, bleached, short-fibered pulp of deciduous trees.

3. The use according to claim 1 or 2, wherein the short-fibered pulp is selected from pulp of eucalyptus, acacia, poplar, aspen, alder and birch.

4. The use according to any of claims 1 to 3, wherein the specific formation index (Sfi) of the paper membrane made is about 0.2 to 1.2, for example about 0.40 Vg/m² ± 20 %.

5. The use according to any of claim 1 to 4, wherein the paper has been provided with detection molecules by at least one method selected from the group of gravure, flexo, inkjet or screen printing, coating and soaking.

6. The use according to claim 5, wherein the detection area has been patterned optionally in a decorative way and optionally combined with printed text or graphics or a combination thereof.

7. The use according to any of claims 1 to 6, wherein the paper or pulp from which the paper has been manufactured is treated with a cellulose ether, such as carboxymethyl cellulose, to modify its capability of wicking liquids.

8. The use according to any of claims 1 to 7, wherein the paper is manufactured from a never-dried or once dried pulp.

9. The use according to any of claims 1 to 8, wherein the porosity and lateral flow capability of the paper membrane is modified by adding a surfactant, for example selected from polyvinyl alcohol and polyoxy alcohol or combinations thereof, or with a polysaccharide, such as starch or starch derivatives, which causes changes to the hydrophilic properties of paper membrane.

10. The use according to any of claims 1 to 9, wherein the paper has been produced by modifying the intensity of wet pressing, e.g. by impingement drying, in order to modify the porosity of the paper membrane.

11. The use according to any of claims 1 to 10, wherein the functionality of the fibrous structure of the paper membrane is modified, e.g. by activating hydroxyl groups of cellulose, in order to increase immobilization of analytes such as proteins to the fibrous network.

12. The use according to claim 11, wherein carbodiimide (CDI) and periodate has been used to activate hydroxyl groups of cellulose.

13. The use according to any of claims 1 to 12, wherein the wet strength properties of the paper membranes was modified by using wet strength chemicals, in particular chemicals selected from the group of polyamideamine-epichlorohydrin (PAE) resin and

ureaformaldehyde (UF) resin and combinations thereof.

14. The use according to claim 13, wherein PAE resin was dosed at a minimum of 1 up to a mximum of about 20 g/kg fibrous matter, in particular about 2 to 5 g/kg fibrous matter, for example about 2.5 g/kg fibrous matter, and alternatively or additionally UF resin was does at a minimum of 1 to 50 g/kg fibrous metier, in particular about 2 to 30 g/kg fibrous matter, in particular about 5 to 20 g/kg fibrous matter, for example about 10 g/kg fibrous matter.

15. The use according to any of claims 1 to 13, wherein the pulp has been produced by an alkaline pulping method, such as sulphate pulping, and bleached with a bleaching process using bleaching chemicals selected from the group of chlorine dioxide, peroxide chemicals and ozone and combinations thereof.

16. The use according to any of claims 1 to 14, wherein the membrane strip comprises lkan absorption pad formed by a portion of the paper strip which is embossed or folded to increase the absorption capacity of the strip.

17. The use according to claim 16, wherein the absorption pad comprises transversal paper strip folding to provide a plurality of adjacent paper layers.

18. The use according to any of the preceding claims, wherein the paper exhibits a combination of the following properties:

- a specific formation index (Sfi) of the paper membrane (handsheet) of about 0.40

- capillary flow time, water, of about 300 s/40 mm ± 25 %; and

- wet strength, measured by a time to break test, greater than that of a nitrocellulose strip having a grammage of 39 g/m².

19. The use according to any of claims 1 to 18 in a laminar flow assay assembly, wherein the paper strip material is being used in the sample pad, the conjugate pad and the absorbent pad of the test strip assembly.

20. A lateral flow assay test strip comprising, in the flow direction, a selectively activated membrane for a test reaction and an absorption pad, wherein

both the selectively activated membrane and the absorption pad are made from a paper strip material, the paper being produced from bleached, short-fibered pulp of deciduous tree.

21. The lateral flow assay test strip according to claim 20, wherein the absorption pad comprises portions of the paper strip embossed and/or folded to increase the strip^'s absorption capacity.

22. A lateral flow assay test strip according to claim 20 or 21, wherein the membrane and the absorption pad comprise an integral paper strip.

23. A lateral flow assay test strip according to any of claims 20 to 22, wherein the absorption pad comprises a protion wherein the paper strip is folded transversally to exhibit a plurality of adjacent and optionally overlapping layers.