Intrant ELISA: A Novel Approach to Fabrication of Electrospun Fiber Mat-Assisted Biosensor Platforms and Their Integration within Standard Analytical Well Plates

Abstract

A combination of far-field electrospinning (FFES) and free-radical polymerization has been used to fabricate coated electrospun polymer fiber mats as a new type of biosensor platform. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) electrospun fibers were dip-coated with different compositions of poly methyl methacrylate-co-methacrylic acid (poly(MMA-co-MAA)). This synergistic approach utilizes large specific surface area of PHBV fibers and co-polymer coatings that feature an optimum concentration of surface carboxyl (-COOH) groups. The platform surface morphology, porosity and tunable hydrophobicity enhance biomolecular interactions via plurality of molecular forces. These customized fiber mats have been integrated into a newly designed 96-well plate called an "intrant enzyme-linked immunosorbent assay" or i-ELISA. I-ELISA allows colorimetric sandwich assay to be carried out without any modifications or additional steps in ELISA methodology. By introducing the fiber mats in fabrication of i-ELISA via extensions on the lid, we address some of the limitations of the previous designs while demonstrating an enhanced signal intensity up to 12 times higher than that of conventional assays. With improved sensitivity, specificity and accuracy in the detection of dengue virus, i-ELISA has proven to be a reliable platform for biomolecular recognition. The proposed fiber mat-assisted well plate in this study holds great potential as a universal approach for integration of different types of fiber mats with pre-designed specific properties in order to enhance the detection sensitivity of the assay.

Paper and Fiber-Based Bio-Diagnostic Platforms: Current Challenges and Future Needs

“…Wax printing [60][61][62][63][64] Whatmann CHR # 1 Assembly of paper in pre-cut lamination sheets [65] PHB fibers Electrospinning and dip-coating with poly(MMA-co-MAA) [5] PHBV fibers [6] Nylon Dip-coating with poly(MMA-co-MAA) [37] Cellulose Photolithography [66] Filter paper Whatman # 42 Commercialized material with proprietary fabrication method~1 cm diameter circles cut into pendent disk shapes to avoid cross contamination [67] Whatman Fusion 5 TM paper Square shaped layers with surface area of 3 cm 2 [68,69] NC Wax printing µPAD with printed channels that function as timing valves [70] Inkjet printing [71] Whatmann CHR # 1 Flexographic printing Polystyrene printed paper (297 mm × 105 mm) with hydrophobic barriers [22] Polyester-backed paper, Whatmann CHR # 1, NC membrane Computer-controlled knife cutting Star, candelabra, and other structures [23] Aluminum foil-baked Whatmann CHR # 1 CO 2 laser cutting/engraving µPAD design with very small features and narrow barriers [72] Whatman CHR # 1 coated with functional polymers/copolymers Vapor-phase polymer deposition Integration of multiple advanced unit operations onto a single µPAD device [73] Whatman # 4 loaded with PEI microcapsules, BCIN, and NBT Suspension mixing followed by filtration Paper strips [74] Nylon # 6 fibers blended with PSBMA and PAA Electrospinning blended fiber deposition on a glass slip [50] PVDF nano-fiber membrane Western blotting platforms (6 cm × 8 cm) [52] Nylon # 6 fibers with Cu-Au nanoparticles Paper strips [75] PCL fiber membrane Folded and pressed sheet of membrane (25 mm × 40 mm) [51] Nomenclature: PHB = Poly(3-hydroxybutyrate; poly(MMA-co-MAA) = poly methylmetacrylate-co-methacrylic acid PHBV = Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); NC = nitrocellulose; PEI = poly(ethyleneimine); BCIN = 5-bromo-4-chloro-3-indolyl-α-D-N-acetylneuraminic acid; NTB = nitro blue tetrazolium; PVDF = Polyvinylidene fluoride; Cu-Au: Copper-gold; PCL = Poly(ε-caprolactone). CHR = chromatography.…”

“…Paper segments in most of the developed platforms are selected from commercially available products (Table 1) possessing certain physical characteristics, including capillary flow rate, surface area, porosity, permeability, and wettability. However, these parameters vary significantly from one manufacturer/batch to another, raising the specter of inconsistency [6,78,79].…”

“…Although relative hydrophilicity and porosity are desirable features in paper-based devices, as they involve capillary forces for leading fluids in specific directions, those characteristics may also introduce background signal and error into the assay [5,6,37,89]. A high degree of hydrophilicity and porosity are not always favorable for protein immobilization and the subsequent detection of analytes as they may induce biomolecule entrapment, which leads to a false positive signal [5,6,90].…”

“…Analytical applications of paper in science date to the early 17th century with the use of cellulose papers for chromatographic purposes [6] and pH sensing [7,8]. A paper-based dipstick for quantifying glucose in urine was first introduced to the scientific community in the 1950s, followed by commercialization of the product for a diabetes test one decade later [9].…”

“…Fabrication of these platforms involves a wide variety of techniques, including plotting [13,14], wax-printing [15][16][17][18][19], inkjet-printing [20,21], flexographic printing [22], computer-controlled knife cutting [23], laser cutting [24,25], vapor-phase polymer deposition [26][27][28], photolithography [29][30][31][32][33][34], spraying [35], electrospinning [5,6,36] and coating [37], among others (Table 1) [2,21]. Different aspects of paper-based bio-diagnostic devices, including fabrication strategies, applications for the recognition of various biomolecular entities, the storability as well as the marketability of the paper-based products have been extensively reviewed [2,3,21,[38][39][40][41].…”