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Chapter 2.7. Adsorptive membranes for diagnostic kit assemblies

Authors: 

Abdul Latif Ahmad, Norhidayah Ideris, Asma Ismail and Low Siew Chun

Art work
Title: 
In a small village
Artist: 
Reiner Kwiotek
Climatic conditions are associated to the increase
 of water-borne and vector transmitted diseases
Looking at faces of people, 
one gets the feeling there's a lot of work to be done.
Wole Soyinka 

Introduction

A biomaterial is defined as a material that can be placed in contact with biological fluids and/or living systems and can be expected to function with a desirable host response in specific applications without exerting any undesirable effects. The use of synthetic polymeric membranes as selective biomaterials has gained increasing attention owing to their versatility and biocompatibility [1]. Moreover, most membrane processes occur at relatively low temperatures and pressures involving no phase changes or chemical additives, and there is therefore minimal denaturation and degradation of the biological products [2]. Several polymer membranes have been used as biomaterials in the separation and purification of biological products. For example, hemodialysis is used to replace some of the functions performed by the kidney, including the elimination of waste products and the restoration of electrolyte and pH levels. Another example is guided bone regeneration, in which a porous membrane acts as a physical barrier to allow a particular tissue to regenerate.

Recently, there has been an increasing interest in the use of polymeric membranes in biosensor applications such as medical diagnostics and environmental pollution control. A biosensor contains a biological entity (such as an enzyme, antibody, or bacteria), which acts as a recognition agent capable of providing quantitative or semi-quantitative analysis [3]. When used in these applications, the membrane is referred to as an adsorptive membrane [2], in which the biological entity is fixed on the membrane surface and acts as a ligand to capture the compound(s) of interest; examples include antibody–antigen diagnostics, species-specific DNA probes, and affinity membrane chromatography. This type of membrane is also known as an assay capture membrane matrix [4]. The interaction between the receptor molecule and the membrane may arise from simple physical entrapment or covalent bonding between recognition elements [5]. Notably, the use of adsorptive membranes has proved very successful in environmental pollution control aimed at the removal of heavy metals such as nickel, cadmium, and arsenic from water sources. For this application, the adsorptive membrane must have the following properties: high abrasion resistance, low surface energy, high surface capacity (for optimal ligand binding), and high mechanical stability. Poly(vinylidene fluoride) (PVDF) and modified chitosan or cellulose acetate have shown great potential as polymers for the development of adsorptive membranes [6, 7].

Membrane preparation

The preparation of synthetic polymeric membranes can be carried out using a variety of methods such as template leaching, stretching, tracks etching, sintering, and phase inversion [8, 9]. However, over the past 20 years, attention has been focused on the use of the phase inversion method owing to its versatility and ability to produce a wide range of membranes. The phase inversion method was developed on the basis of the differing affinity of a solvent towards the coagulation material and phase. In this process, a polymer solution is cast in the desired membrane shape and subsequently brought into contact with a non-solvent (commonly called the coagulant), which is miscible with the casting solvent [10].

There are two types of membrane, which are distinguished by their structures: symmetric or asymmetric. This difference occurs during the polymer fabrication process and the choice of polymer used, based on the future application of the membrane. An adsorptive membrane requires a symmetric, microporous structure. Determination of the symmetric or asymmetric structure is mostly based on the mass transfer process between the solvent and the coagulants, and the thermodynamic equilibrium between the two; this significantly affects the membrane morphology and pore size [10]. The phase inversion process covers three commonly used techniques: thermally induced phase inversion (TIPS), vapor-induced phase separation (VIPS), and non–solvent-induced phase separation (NIPS) [8, 10].

Thermally induced phase separation

The demixing of a polymer solution in a mixed or single solvent is achieved by cooling the polymer solution to below the critical temperature to enable phase separation [11, 12]. In the TIPS process, the selection of solvent is an important factor for controlling the membrane morphology and directly influences the thermodynamic properties of the polymer-solvent system [13].

Vapor induced phase separation

In a typical VIPS process, the cast film is exposed to humid air prior to immersion in the coagulation bath. The exposure to humid air induces polymer precipitation by transferring the non-solvent from the gas phase to the cast film [11]. Water is commonly used as the non-solvent in the VIPS process, and water transfer arising from the exposure to humid air leads to phase separation. Other factors that influence the VIPS technique are: air velocity, relative humidity, air temperature, and exposure time [14].

Non–solvent induced phase separation

Immersion precipitation by a non-solvent or non-solvent phase separation is the most commonly reported techniques used in the phase inversion process. Phase separation occurs when a clear dope solution consisting of a polymer and a solvent (or sometimes a non-solvent) is formed into the desired membrane module (such as a flat sheet or hollow fiber), and subsequently contacted with a non-solvent, which is miscible with the solvent. The transfer between solvent and non-solvent leads to phase inversion and the formation of a three-dimensional porous polymer network. This technique is characterized by a rapid mass transfer rate resulting in a dense layer on the membrane surface [14]. However, the mass transfer rate can be controlled by manipulating the immersion time, and the composition and temperature of the coagulation bath.

The structures of the membranes produced are also influenced by other factors including the composition of the casting solution, the choice of polymer, solvent, non-solvent and additives, and the gelation and crystallization behaviours of the polymer formed. The conditions used in the casting process also influence the membrane structure with factors such as initial casting thickness, casting speed, temperature, and humidity [12]. For example, an adsorptive membrane designed for use and application in a diagnostic kit requires careful selection of the casting thickness to ensure that the desired morphology and mechanical strength are incorporated for ease of use and handling. By carefully selecting the membrane fabrication technique, and the formulation and casting process conditions, the membrane morphology can be controlled, and porous membranes can be prepared at the desired pore size, porosity, and yield thickness.

Membrane properties for application in diagnostic industry

Advances in modern medical technology have played a significant role in human medicine by improving the prevention and treatment of disease. A combination of medical expertise and appropriate medical facilities is required to ensure the successful delivery of medical treatments to patients. However, the annual rate of mortality is very high, especially in under-developed and developing countries. This is mainly attributed to the scarcity of medical resources, such as highly trained specialists and advanced diagnostic equipment [15]. Medical technologies are usually only available in urban areas and are therefore in great demand, which results in long waiting times for access. Hence, to address these issues, sensitive, affordable, and efficient diagnostic tool kits are highly desirable to assist medical workers and offset the unavailability of non-portable, high-end technology in rural areas.

Usually, diagnostic tool kits are functionally based on the concept of immunoassays, such as those used in the initial screening for HIV [16, 17], tuberculosis [18], typhoid [19], and pregnancy [20]. Immunoassays are antibody-based detection systems for specific antigens, and their efficiency originates from the substrate specificity of an antibody for a particular antigen. The assays function by measuring the concentration of a substance in a complex biological liquid system, typically blood serum or urine. In a homogenous immunoassay, the antigen, antibody, and sample are mixed in the solution phase. In contrast, in a heterogeneous immunoassay, one constituent is immobilized on a solid surface and the other constituents are delivered in the solution phase [21, 22]. A heterogeneous assay is an attractive choice for overcoming the limitation of very low concentrations of biological markers in bodily fluids. For this application, the immunoagents are immobilized on a polymer membrane matrix in a process known as protein immobilization. In these systems, an immobilized antigen detects an antibody or an immobilized antibody detects an antigen [3].

For detection, the immunochemical reaction can be visualized using enzyme labels, radioimmunoassays, fluorescence polarization, chemiluminescence, and electrochemical methods, based on the specific needs of the test itself. Immunochemical-based assays are routinely used in diagnostic kits for environmental and food testing (immunoassay and immunosensor) and separations (immunoaffinity chromatography) [23]. However, amongst these immunochemical analytical methods, the immunoassay is the most utilized (especially in the field of medical diagnostics) owing to its operational simplicity and speed, even if no information on the size of the target biomolecule is provided. Quantitative or semi-quantitative analysis can be provided using a choice of detection methods based on electrochemical, mass, thermal, or optical properties [5].

A well-defined adsorptive membrane is required for optimum protein immobilization. Ideal materials have stable and controllable surface properties, a large surface area, high binding affinity and stability with regard to biomolecules, and favorable handling properties; they should also be cheap to manufacture by large-scale production processes [24]. Inorganic materials such as metals and ceramics have been used as materials for solid phase applications because they have versatile electrical properties that can be effectively and easily modified. However, such materials are often costly because they are naturally scarce and expensive to manufacture. Therefore, synthetic porous polymer membranes are a preferred alternative because they are inexpensive, easy to process, and have a high surface area [25].

Membranes with different pore structures or morphologies have been used because specialized membrane materials, surface properties, structures, and membrane dimensions have been required for different reagents in various downstream processes. Thus, the determination of the membrane characteristics, especially the morphological characteristics (such as symmetry, pore radius, pore volume (porosity) and co-continuity of pores) is very important for optimal protein binding. In general, a combination of small pore size and high porosity is required to obtain a microporous membrane with high interconnection between the pores [26]. Such morphology is desirable in a potential capture reagent because it provides accessibility and a large surface area for potential protein immobilization.

In general, a protein can bind onto a membrane surface by one of several mechanisms: hydrophobic interactions, electrostatic interactions, hydrogen bonding, or van der Waals interactions. Van der Waals interactions are a combination of several types of very weak, short-range bonding interactions including dipole–dipole, induced dipole–induced dipole, and dipole–induced dipole interactions. These interactions are weak and difficult to control through the choice of experimental conditions. Hydrogen bonds, a special type of dipole–dipole interaction, are stronger and play important roles in protein folding and adsorption. However, hydrophobic and electrostatic interactions are the most important driving force for the adsorption of protein on a membrane surface. Hydrophobic bonding interactions are a result of the dehydration of the apolar parts of the protein and the membrane, whereas electrostatic bonding interactions arise from Coulombic attraction or repulsion between charged groups. Electrostatic effects are observed when the protein and membrane surface have opposite charges, and can be influenced by dilution or changing the pH of the system. Electrostatic interactions are usually relevant on hydrophilic membranes or sorbent surfaces; when repulsive, they can prevent protein immobilization [27, 28]. However, on a hydrophobic surface (such as that found on a PVDF membrane), the hydrophobic interaction is dominant, facilitating high protein binding even under electrostatically adverse conditions [29].

The pore structure of a membrane is crucial for the accuracy and sensitivity of an immunoassay. Membranes with lower pore sizes exhibit enhanced protein immobilization in the membrane reaction zone, producing a more sensitive assay. The immobilized protein can bind firmly and diffuse horizontally within a smaller pore matrix because there are more interconnecting polymer structures within the membrane. A smaller pore size also creates higher diffusion resistance to the liquid flow in the membrane after protein deposition. The slower wicking rate increases the duration that the protein interacts with the polymer membrane matrix [30]. In contrast, larger pore sizes with fewer interconnecting polymers in their structure reduce membrane–protein interactions and cause the protein to detach during the washing process. Moreover, a less interconnected polymer matrix causes the deposited protein solution to diffuse vertically owing to gravity and reduces protein retention on the surface of the membrane.

If the working mechanism of the developed diagnostic is lateral flow, the lateral wicking rate must be taken into consideration. In the application of a lateral flow diagnostic strip, an optimal combination of fast lateral wicking rate and high protein immobilization is desirable to produce efficient diagnostic performance. In such a case, other factors including porosity, pore connectivity, and tortuosity can have a significant effect on diagnostic performance because the protein solution diffuses both vertically (during the protein immobilization) and horizontally (from the target analyte to the point of deposition). Ahmad et al. reported that the membrane wicking time was governed by both the morphology (pore size and porosity) and the properties of the individual additives included during membrane fabrication [31].

Membranes for protein immobilization

Nitrocellulose (NC)

A number of polymeric materials have been used to improve final assay performance and membrane function by manipulation of the material properties during the fabrication process. Historically, the most frequently used membranes in immunoassay applications have been made from NC [32]. NC, also known as Type ‘RS’ or Type ‘E’ (11.8–12.3% N), is a well-known polymer that is used to produce adsorptive membranes with excellent characteristics such as high porosity, high pore connectivity, and high binding affinity. In terms of its polymer chemistry, NC is categorized as a Lewis acid, is soluble in a wide range of inexpensive organic solvents (such as acetone and methyl acetate), and can be manufactured with pore sizes ranging from 0.05 to 12 µm [33]. An NC membrane is inherently hydrophilic, which makes it easy to use because a solution of protein can be immobilized directly on its surface without any pre-treatment [30, 34].

Moreover, the ability of NC to bind protein is commonly accepted as a universal property of these membranes. The NC membrane–protein binding mechanism is attributed to the strong dipole of the nitrate group present in the membrane interacting with the strong dipoles present in the peptide bonds of the bound protein, giving rise to electrostatic affinity [34-36]. As mentioned earlier, electrostatic interactions on hydrophilic membranes or sorbent surfaces are important. Thus, the mechanism of protein binding on the NC membrane is influenced by changes in the dilution and/or pH of the protein solution. The use of NC membranes has been reported in a variety of studies including those investigating the detection of infectious diseases [37, 38] and food hygiene [39, 40]. Although NC is the most commonly documented membrane material in protein immobilization, its applications are limited because it has low resistance to harsh conditions such as those found in highly acidic and alkaline environments. Moreover, NC membranes become brittle and discolored through use, a property not observed with analogous membranes constructed from nylon, polysulfone, or certain acrylic copolymers [41].

Poly(vinylidenefluoride) (PVDF)

To overcome the weak mechanical characteristics of NC, research has led to the development of alternative types of polymer membranes. PVDF displays excellent mechanical characteristics compared with NC. PVDF is a semi-crystalline polymer obtained by the polymerization of vinylidene fluoride monomers. Fluoropolymers have increased thermal stability compared with their hydrocarbon equivalents. This stability is attributed to the high electronegativity of the fluorine atoms present on the polymer chain and the high bond dissociation energy of the C-F bond [42]. PVDF is a commercially available polymeric material with several desirable properties including high thermal stability, low surface energy, and good physicochemical properties [43]. Aizawa and Gantt have shown that PVDF has great potential as an adsorptive membrane owing to its high level of non-specific bonding interactions with proteins [44]. Many reports have described the application of PVDF membranes as capture reagents in various immunoassays including the detection of mycotoxins, mouse IgG, and proteins [44-46].

Recently, there has been an increased use of PVDF in biomedical applications because it has several advantages over other hydrophobic membrane materials. Generally, hydrophobic materials increase adsorption by promoting irreversible hydrophobic bonding interactions between the membrane and the protein. As mentioned earlier, protein–membrane interactions are largely the result of physical adsorption arising from electrostatic and hydrophobic interaction [29]. PVDF membrane matrices are strong and have high protein-binding capacity. Moreover, PVDF is inert and is not easily extracted; this makes it a suitable candidate for biomedical and bio-separation applications because it promotes the non-specific adsorption of proteins [47]. PVDF is also known for its piezoelectric properties, which are attributed to its semi-crystalline structure. Its piezoelectric properties are approximately 10 times greater than those observed in other polymers, making it a valuable material for sensor applications [48]. The hydrophobicity of PVDF is advantageous because it promotes irreversible protein–membrane binding interactions. However, PVDF membranes require pre-wetting before protein immobilization, which can be time-consuming and can hinder protein immobilization.

Nylon

Along with NC and PVDF, the other commonly used polymer membrane in protein immobilization is nylon. Nylon is composed of repeating sub-units linked by peptide bonds, and is frequently referred to as a polyamide. Nylon has great potential in the development of adsorptive membranes because it has good functional properties such as consistent pore size distribution, excellent mechanical resistance, and flexibility without the need of a plasticizer [49]. Nylon is also hydrophilic in nature, and is compatible with a range of aqueous and alcoholic solvents; it is cationic and maintains its positive charge over a wide pH range [50]. The charge characteristics of nylon make it an effective and easily modified adsorptive membrane material that can provide stable binding through physical adsorption and covalent bonding interactions.

Examples of the use of nylon as an adsorptive membrane are in the detection of antigen–antibody complexes [51], species-specific DNA probes [52-54], and specific biomolecules via the immobilization of enzymes [55, 56]. However, the major drawback in the use of nylon as a membrane is the high staining background observed owing to strong charge interactions, making it unsuitable for protein immunoblotting with detection methods based on the color development of a chromogenic reactant. Furthermore, the piezoelectric properties of nylon remain a challenge in the presence of hydrogen bonding , and water absorption can have a deleterious effect on its dielectric and piezoelectric properties [57]. Table 1 summarizes and compares the characteristics of the three types of polymeric membranes commonly used in protein immobilization. The choice of a particular membrane depends on the particular requirements of its application.

Table 1. Summary of the characteristics of polymeric membranes.

Working mechanisms of membrane

Lateral flow

Lateral flow orientation is one of the working mechanisms used in immunoassay applications. Under this orientation, the two most relevant parameters that define the performance of the membrane are: (1) its ability to bind proteins, and (2) the lateral diffusion rate of the protein along the horizontal axis. In an immunoassay, the lateral flow membrane carries the target analyte along the membrane strip and binds it to the immobilized capture reagent in the membrane capture zone [67]. The solution containing the target analyte (either antigen or antibody) is moved by capillary force along the narrow rectangular membrane strip. When the analyte moves along this flow path, it comes into contact with the immobilized capture reagent that has been previously deposited and dried on the membrane capture zone [67]. The target analyte is separated from the sample solution when it binds to the immobilized capture reagent. After washing, the test result is clearly visible owing to the appearance of a colored band on the membrane strip. A summary of the basic working mechanism for lateral flow is shown in Figure 1.

Figure 1. Basic principle employed by a diagnostic test strip using lateral flow orientation.

If a membrane has good binding properties, the capture line initially deposited on the membrane is not easily washed away by the capillary forces of the buffer solution. A short lateral wicking time causes the target analyte to diffuse faster to the point of interest resulting in rapid detection.

Dot-blot

The dot-blot technique requires the early separation and purification of the target protein (antigen or antibody), commonly using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel electroelution [68]. In the dot-blot technique, a sheet of membrane matrix is used. Subsequently, either the antigen or antibody is spotted directly onto the membrane. Monoclonal antibodies are often used because they usually selectively bind to a particular molecule to provide a more accurate result. The sensing route of the dot-blot is the same as that used for all immunoassay techniques, and is often based on colorimetry, fluorescence, or chemiluminescence. Dot-blots have become a preferred choice for use in immunoassays because they are less time-consuming and more cost effective, accurate, and simple to use [45]. Dot-blotting is also suitable for large-scale field screening. Figure 2 summarizes the flow process in the dot-blot technique with colorimetry as the detection method. In this workflow, the antigen is considered to be the capture reagent and is immobilized on the membrane surface.

Figure 2. Dot blot working mechanism

Conclusion

Based on the review presented, it is clear that polymeric membranes have great potential in the development of diagnostic kits. For enhanced performance in diagnosis, certain membrane characteristics are required to ensure the sensitivity and reliability of the test. The morphology of the membrane can be controlled, and a porous, symmetric membrane can be prepared with the desired pore size, porosity, and yield thickness by careful selection of the fabrication technique, the formulation, and the casting conditions. In terms of membrane morphology, a combination of small pore size and high porosity is required for a microporous membrane with high interconnection between pores, which offers good accessibility and a large surface area for potential protein immobilization. Moreover, the materials used for adsorptive membranes should have other characteristics such as stable and controllable surface properties, a large surface area, high binding affinity and stability with regard to biomolecules, and favorable handling properties; they should also be cheap to manufacture. Among the several types of polymers reported, NC, PVDF, and nylon are the most commonly used in membranes for diagnostic kit assemblies.

Acknowledgments

The authors are grateful for the financial support granted by Universiti Sains Malaysia (USM) for the Vice-Chancellor Award 2010 and MTDC Grant (6053014/M130) sponsored by the Malaysia Technology Development Corporation. All authors are affiliated with the Membrane Science and Technology Cluster of USM (8610012)

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