PHYSICOCHEMICAL MODIFICATION AND APPLICATION OF ALGINATE GELS FOR THE CONTROLLED RELEASE OF REAGENTS IN CLASSICAL SOLUTION ASSAYS AND MICROFLUIDIC ASSAYS

Abstract

A reagent assay having a tubular member having a cross-section. The assay further has a first porous alginate plug shaped having the cross-section within the tubular member and containing a first reagent and a second porous alginate plug shaped having the cross-section adjacent the first porous alginate plug within the tubular member and containing a second reagent.

Claims

1. A reagent assay, comprising: a tubular member having a cross-section; a first porous alginate plug shaped having the cross-section within the tubular member and containing a first reagent; and a second porous alginate plug shaped having the cross-section adjacent the first porous alginate plug within the tubular member and containing a second reagent.

2. A method of modifying alginate, comprising: providing a sample of porous alginate; and mixing in the alginate a reagent that is encapsulated by one or more pores in the alginate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The various embodiments of the present disclosure are best understood by referring to FIGS. 1 through 6 of the drawings. The elements of the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the disclosure. Throughout the drawings, like numerals are used for like and corresponding parts of the various drawings.

[0008] FIG. 1 is a representation of the molecular make-up of alginate.

[0009] FIG. 2 is a graph showing absorbance versus wavelength of various gel and gum materials.

[0010] FIG. 3 depicts a prepared bead for reagent assay in accordance with an embodiment of the present disclosure.

[0011] FIG. 4 depicts enhanced porosity of alginate in alginate samples.

[0012] FIG. 5 depicts another reagent assay in accordance with another embodiment of the present disclosure.

[0013] FIG. 6 depicts an alginate lyase.

DETAILED DESCRIPTION

[0014] The present disclosure describes a composition of a porous alginate and method of chemically modifying the porosity and conductivity of alginate that can in tandem with other degradable polysaccharides be used in the controlled release of reagents for chemical assays. By modification of alginate with certain solubilized ionic salts, various degrees of porosity can be generated in the hydrogel. The porous hydrogel may be used as a filtering device. By addition of solutions of electrolytes and oxides the conductivity of the alginate hydrogel can be increased or decreased, respectively. The porous alginate can incorporate a reagent used for the determination of an analyte. The reagent releases on exposure to the reacting solution. The porous alginate can be used as a hydrogel core for a series of coatings of reagents mixed with various molecular weight polysaccharides. These are designed to release the reagents serially from the outer layer to subsequent interior and finally inner hydrogel core layers, based on the decreasing solubility of each polysaccharide. If the analyte is sensitive to dilution, the selected polysaccharide(s) can be solubilized with the specific affiliated lyase. The catalytic mechanism, via the breaking of glycosidic bonds in the polysaccharides increases the solubility with negligible dilution of the analyte sample. Alginate hydrogel has two affiliated lyases. This enables implantation of toxic or catalytic materials in the hydrogel, minimizing exposure of technical personnel while permitting recycling and recovery of the catalytic material at an appropriate facility.

[0015] Alginate, once gelled does not solubilize on further dilution with the same solvent (in contrast to the other mentioned high molecular weight polysaccharides that dissolve in excess solvent). Alginate can have gel setting times of 1-20 minutes and this is a function of the salt used, the dilution factor and set temperature. For the purpose of designing alginate of varying porosity a longer setting time of at least 10 minutes is desirable. To achieve the desired setting time a dilution factor of 1:5 parts (dry weight of alginate plus added reagents to solvent weight of distilled water). This enabled homogeneous mixing of the gel during the workable stage to a smoother consistency and time for pipetting of various volume droplets (in these experiments 50-300 microliter beads of initial volume were produced) onto Teflon baking sheets or wax paper prior to being fully set. This mixing and droplet (bead) forming process can be easily automated with existing industry equipment. Alternately larger gel samples were prepared by placing the samples in 1 mL or 5 mL pipet tips. Once fully set alginate undergoes syneresis. This is the process that sparked interest in the investigation of alginate for reagent delivery and led to subsequent other discoveries and applications. Syneresis is the expulsion of liquid from the set gel as it undergoes contraction. The gelled droplets sweat.

[0016] The first experiment performed was the coating of prepared beads using the syneresized liquid from the bead to adhere a dry chemical reagent. It was found that further evaporative loss could be prevented by dusting the reagent covered bead with confectioner sugar. Further investigation demonstrated that other gum types were more suitable and stable as a coating seal material. These included, but are not limited to, gum arabic, locust bean gum, guar gum, carrageenan and gellan. UV Spectrophotometric analysis, a graph of which is shown in FIG. 2, of these polysaccharides showed that the absorbance of these species was below 310 nanometers (nm) (in all samples except guar gum that continued to show slight absorbance between 0.075 decreasing to 0.015 at 390 nm) and therefore insignificant and well below the range of observation for the testing of drinking and waste water qualitythe first target applications.

[0017] A second series of experiments were performed that demonstrated that a second reagent could be added to the first reagent over the alginate core, by mixing it in gellan and applying by a rolling process to the existing bead. This could be done in a manufacturing process through a spray coating and drying process.

[0018] A third series of experiments demonstrated that by approaching the limit of gelation of alginate (higher ionic strength addition), in addition to protracting the setup time, the alginate had greater porosity. By mixing the added salts (source of cations) with high molecular weight dyesspecifically Allure (a red dye) and Blue Dextran at these higher ionic strength solutions, it was found that the alginate matrix at the selected ionic strength did not retain these dyes. However in samples mixed with lower concentration of salt, these dyes were entrapped within the resulting alginate hydrogel. The ionic strength desired to obtain high pore content were able to be visually determined by the included indicator dye. When alginate at the specific ionic strength was mixed with blue or red dye to produce a pale colored hydrogel, a highly porous gel matrix was indicated when a concentrated dye exudate appeared during syneresis. If the ionic strength was lower, the gels retained the pale coloration as the dye was entrapped within the gel. Thus, to produce higher porosity of the alginate was experimentally determined to be at the limit that maintained larger gel pores capable of exuding the bulky dye molecules. Upon soaking in solvent the entrapped dye in the lower ionic strength gel mixtures eventually diffused from the colloid indicating that the retention time of the dye was very protracted due to smaller pore size.

[0019] Disclosed is a method of chemically modifying the porosity as depicted in FIG. 4 and that can in tandem with other degradable polysaccharides be used in the controlled release of reagents for chemical assays as shown in FIG. 3. By modification of alginate with certain solubilized ionic salts, various degrees of porosity can be generated in the hydrogel. The hydrogel formulation contained 92% to 98% weight to total weight (% w/w) of dry sodium alginate with up to 8% added salt. Note that the percentages of substances is for exemplary purposes and other percentages of substances may be used in other embodiments. This range reflects the use of two product sources of sodium alginate. One source is a prosthetic grade sodium alginate, a product from harvested brown seaweed (kelp), and the other source was Reagent Grade Alginic Acid Sodium Salt by ACROS (CAS 9005-38-3Lot A0339825). A matrix screen was conducted with various additions of sodium, calcium, or potassium salts at ambient temperature (68 F./20 C.) or slightly below ambient temperature. The result of the matrix screen indicated that the potassium cation from potassium chloride (KCl from Fisher Scientific 525484A Lot 4AH1307-3125A) and potassium iodide (KI from ACROS CAS 7681-11-0 Lot B0137921) produced large pores that retained uniformity. The amount of the potassium iodide required for the pore formation was 4% w/w when using the prosthetic grade alginate as depicted in FIGS. 4. 400 and 401 (repeat sampling) that shows uniformity in the matrix screen. The ACROS product also produced uniform pore formation of comparable size at 7.5% w/w. Greater addition of the selected salts were needed when using the ACROS product or Fisher product, presumably as a result of product higher purity. The alginate once gelled remained stable and pliable for several months when stored in a container of minimal air volume to maintain humidity level. This enables storage of prepared beads.

[0020] FIG. 3 depicts a prepared bead 300 in accordance with an embodiment of the present disclosure. The prepared bead 300 comprises an inner porous alginate core 301 created by modification of alginate hydrogel, as described further herein, and containing a first reagent A. Note that the identification of reagent A in the alginate core 301 is for exemplary purposes only, and greater or fewer occurrences of reagent A are possible and probable. The prepared bead 300 shows the porous core on which additional polysaccharide plus reagent layers may be added comprising outwardly extending layers as shown in FIG. 3. The outer layer 304 is solely a polysaccharide used as a seal.

[0021] As noted hereinabove, by modification of alginate hydrogel via inclusion of various percentages by weight (hereinafter % w/w) of sodium, calcium and potassium salts that introduce porosity within the hydrogel, an analyte assay reagent may be incorporated within these pores and subsequent polysaccharide coating layers and be released on exposure to aqueous solution or solvent. Note that the hydrogel formulation contains 92% to 98% weight to total weight (% w/w) of dry prosthetic grade sodium alginate. The prepared salts of alginic acid (sodium alginate, potassium alginate or ammonium alginate) form an irreversible hydrocolloid gel matrix in comparison to the non-water soluble alginic acid. Therefore the prepared salts are the base formulation used in these compositions prior to addition of further salts used to generate the pore formation. Addition of the analytical reagents may be mixed into wet pre-set alginate, adhered to the surface of set alginate, incorporated into dehydrated dried porous alginate in reagent solution under vacuum or tumbled as a dry reagent into dry porous alginate beads.

[0022] In accordance with an embodiment of the present disclosure, a serialized timed-release of reagents, e.g., reagents A, B, and C depicted in FIG. 3, can be achieved by encapsulating reagents within and in surrounding layers of a porous hydrogel core, as described hereinabove. As shown, reagents may be layered on the alginate core 301 separated by a series of polysaccharides layers 302 and 303 of successively lower solubility as the layers 302 and 303 move outward from the core. Note that each reagent A, B, and C would be mixed with a gum agent that includes but is not limited to locust bean gum, guar gum, gum arabic, tara gum, xanthan gum, pullulan gum and gellan gum. The molecular weight composition and affiliated solubility of these gums in aqueous solution vary. Reagents are solubilized by exposure to aqueous solution until all of the reagents have reacted in the analyte assay sequence, minimizing the reagent quantities needed, maximizing reaction efficiency and eliminating competing reactions that occur in alternate bulk solution processes. As shown in FIG. 3, the porous core 301 contains the last reagent for release in the analyte process and below the outer polysaccharide seal is the first reagent C to be released. Each of the reagents C, B and A would be released by dissolution of the previous layer.

[0023] In one embodiment, use of lyase 600 as shown in FIG. 6 in the assay reduces the amount of solvent (usually water) to dissolve the polysaccharide-reagent containing layers 302-303 shown in FIG. 3. If dilution by aqueous medium of the analyte is problematic, each of the long chain polymer polysaccharide gums selected can be enzymatically cleaved into smaller segments by lyase 600. Note that lyases are specific for cleavage of a particular polysaccharide (e.g: a lyase for gellan would be different than one for alginate). The mechanism of action is usually through cleavage of the glycosidic bond through a (3-elimination process. By breaking these long chain polymers into small chains, solubility is enhanced. As a catalytic material, lyase addition (usually by droplet) is a small volume addition. As an enzymatic protein catalyst a spectrophotometric peak will be evident at 235 nm. This UV region is usually below the analyte test region for conventional water or wastewater analysishowever it may interfere with certain biological testing assays centering on 235 nm.

[0024] Note that in one embodiment, the porous hydrogel without the inclusion of reagents may be used as a filtering device or size exclusion device in columns, funnels, pipets and microfluidic channels.

[0025] FIG. 5 depicts another exemplary reagent assay 500 in accordance with an embodiment of the present disclosure. The reagent assay 500 comprises a container 505, which may be for example an open-ended pipet. In addition, the reagent assay 500 comprises one or more porous alginate plugs. In this regard, the reagent assay 500 comprises porous alginate plugs 502-504.

[0026] As noted hereinabove, each porous alginate plug 502-504 is made up of a porous alginate substance. Further, within the porous alginate substances are respective reagents A, B, and C. Note that only a single letter, including A, B, and C, are shown for exemplary purposes depicting the reagent. However, each porous alginate substance includes a myriad of instances of the reagent contained therein.

[0027] In use, an analyte solution, depicted by reference arrow 506, is poured into the container 505 in the fluid reservoir 501. From there, the analyte solution 506 travels through the first porous alginate plug 502 reacting with reagent A. Thereafter, the analyte solution 506 travels through porous alginate plug 503 reacting with reagent B, then through porous alginate plug 504 reacting with reagent C. Once the analyte solution 506 has traveled through each porous alginate plug, the reacted analyte solution, depicted by reference arrow 507, may exit the tube for further analysis.

[0028] In such an embodiment, a series of tubular slugs of alginate containing reagents can be aligned and placed in tandem within a cylindrical tube. The analyte can be placed at the opening of the tube and permitted to percolate through each successive slug. The solution exiting the tube (outflow) would then be ready for the detection method for that analyte. This could also be readily adaptable to microfluidic methods where the gelled reagents are placed inline in the microfluidic channels. This enables the sealing of a microfluidic chip with all reagents constrained within an insoluble and immobilized gel. This type of microfluidic chip could function in gravity driven, applied pressure or in sipper mode (pulling of the solution by gentle suction on the outflow port). For utilization of this method in the field, a tube (straw-like) could include the gel slugs with reagents, the analyte, for example a water sample, could be guided gently through the tube (using a syringe bulb) and the sample collected for detection. If the method is colorimetric this could be compared to a color chart.

[0029] Note that in one embodiment the porous alginate core 301 (FIG. 3) may be used as a non-dissolving substrate to contain catalytic materials that may be reused or recovered for recycling. This sustainable method also promotes safety in handling. The alginate core is an irreversible hydrocolloid gel. In processes requiring rare or toxic catalyst inclusion, the ability to recover these materials is important. As an example, cadmium beads within a glass column are often used in reduction assays. Cadmium beads could be mixed with porous alginate, confining it for safety within the gel (breakage of a glass column would not cause cadmium spillage as it would remain intact in the hydrogel). In microfluidic applications where reagents are stored on a chip the catalytic material could be extracted via a punch mechanism and recycled. Alginate also has two specific lyases. One breaks the glycosidic bond of an M-block and the other breaks the glycosidic bond of a G-block. Therefore addition of these lyases to alginate, and subsequent disruption of those bonds enables extraction of the cadmium beads on sedimentation or sedimentation-assisted centrifugation.