A LEGO inspired fiber probe analytical platform for early diagnosis of Dengue fever

Materials Science & Engineering C 109 (2020) 110629

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A LEGO inspired fiber probe analytical platform for early diagnosis of Dengue fever

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Samira Hosseinia,b, , Pedram Azaric,d, Braulio Cardenas-Beniteza, Eduardo Martínez-Guerrae, Francisco S. Aguirre-Tostadoe, Patricia Vázquez-Villegasa, Belinda Pingguan-Murphyc,d, Marc J. Madouf,g, Sergio O. Martinez-Chapaa a

Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, NL 64849, Mexico Writing Lab, TecLabs, Vicerrectoría de Investigación y Transferencia de Tecnología, Tecnologico de Monterrey, Monterrey 64849, NL, Mexico c University of Malaya, Department of Biomedical Engineering, Faculty of Engineering, Kuala Lumpur 50603, Malaysia d University of Malaya, Centre for Applied Biomechanics, Faculty of Engineering, Kuala Lumpur 50603, Malaysia e Centro de Investigación en Materiales Avanzados S. C. (CIMAV-Unidad Monterrey), NL, Mexico f University of California, Department of Biomedical Engineering, Irvine, CA 92697, USA g University of California, Department of Mechanical and Aerospace Engineering, Irvine, CA 92697, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Paper-/fiber-based analytical platforms Dengue detection Surface functional groups Integrated bio-sensing platform Electrospinning

Based on the concept of LEGO toys, a fiber probe analytical platform (FPAP) was developed as a powerful diagnostic tool offering higher sensitivity in detection of infectious agents compared to established methods. Using the form and the function of LEGO toys, this protocol describes a fiber-based, 96-well plate, which suspends a new class of chemically-designed, electrospun fibers within the assay. This clamping strategy allows both sides of the developed fiber mats to interact with biomolecules within the assay thus benefiting from the tailored chemical and physical properties of these fiber-based bioreceptors in attracting the biomolecules to the surface. The fabrication method of FPAP involves one-step electrospinning of the chemically designed fibers, 3D printing of the LEGO-like probing segments, and assembly of the device followed by ELISA procedure. FPAP follows the same principles of operation as that of a conventional enzyme linked immunosorbent assay (ELISA), therefore, it can be run by lab technicians, expert in ELISA. FPAP was used for early diagnosis of Dengue fever and provided an 8-fold higher sensitivity while the limit of detection (LOD) was recorded to be in femto-gram per milliliter range which is significantly low when compared to other existing techniques or conventional assay. This platform allows different types of paper/fiber bio-receptive platforms to be incorporated within the design that promises simultaneous recognition of multiple infectious agents.

1. Introduction Different classes of paper-based analytical platforms have been developed, including dipsticks, lateral flow assays (LFAs), paper-based analytical devices (μPADs), and micro well plates [1–5]. The majority of these platforms have characteristic beneficial features, as they are: (i) rapid and cost-effective; (ii) light weight; (iii) operating with small sample volumes; and (iv) providing naked-eyed detection, among others [6–8]. However, they also suffer from certain limitations, including: (i) insufficient detection sensitivity; (ii) instability of the paper materials; (iii) inertness of the paper that renders a general need for surface treatment; and (iv) brief shelf-life of the platforms, specifically after surface activation [8–10]. Applied papers in most of these devices

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are chosen from commercially available nitrocellulose (NC), which, in turn, might vary in properties from one manufacturer to another [9]. Therefore, there is an obvious need for development of customized paper materials with desirable chemical and physical properties that make the treatment steps unnecessary [9,11]. Polymer-based fibers/papers have garnered considerable attention in the field of bio-diagnostics for development of such customized materials. Electrospinning, in specific, has played a vital role in development of effective bio-receptive platforms [12,13]. Işika et al., reported a Polyvinyl alcohol (PVA)-based electrospun nanofiber for lipase immobilization which has shown enhanced enzyme stability with high degree of protected enzyme activity [13]. Reukov et al., reported nanocoated nylon fibers, with positively charged surfaces designed for

Corresponding author at: Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey, NL 64849, Mexico. E-mail address: [email protected] (S. Hosseini).

https://doi.org/10.1016/j.msec.2020.110629 Received 19 October 2019; Received in revised form 18 December 2019; Accepted 2 January 2020 Available online 07 January 2020 0928-4931/ © 2020 Elsevier B.V. All rights reserved.

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Table 1 Comparison of different platforms for NS1 Dengue detection with their evaluation parameters. ELISA test kit

ELISA protocol

Sensitivity (%)

Specificity (%)

Accuracy (%)

LOD

Ref.

Platelia™ (Biorad Laboratories)

Indirect

Early ELISA (PanBio™)

Indirect

NS1 Ag Strip (Biorad Laboratories) Dengue NS1 Ag ELISA (Standard Diagnostics, Inc) Custom made ELISA Ag-ELISA Ultrasensitive Hetero-sandwich ELISA i-ELISA FPAP

Indirect Indirect Sandwich Sandwich Sandwich Indirect Indirect

93 73 83 – 66 72 89 70 – – – 97 100

100 100 99 – 89 100 99 71–80 – – – 87 93

100 100 98 – 76 100 99 – – – – 94 97

– – – 250 ng/mL – – – – 7 to 284 ng/mL 30.4 pg/mL 31.25 pg/mL 1.4 pg/mL 0.5 pg/mL

[37] [38] [39] [40] [36] [39] [39] [41] [34] [42] [33] [22] Current work

laboratory techniques for DF biorecognition includes viral isolation, viral RNA detection, and specific antibody detection (IgM/IgG) within sera [30,31]. While viral isolation is a costly method and requires appropriate cell culture infrastructure, it typically provides the results after 6 to 10 days. This may pose serious risks since DF could result in patience's death within 14 days. Sophisticated techniques including polymerase chain reaction (PCR) or reverse transcription polymerase chain reaction (RTPCR) provides results within 24 h [32]. These methods are time consuming, expensive, and are not available in every clinical setup [33]. Commercial immunochromatographic and enzyme linked immunosorbent assay (ELISA) provide Dengue diagnosis within few minutes and few hours, respectively. The both methods rely on detection of IgM/IgG antibodies against Dengue which is only possible after 4 to 5 days of the disease [27,32]. Non-structural protein 1 (NS1) circulates in the blood stream since the onset of the fever hence proven to be the right target for early Dengue diagnosis. Concentration of circulating NS1 proteins in blood changes with gender, age, primary or secondary infection (typically higher in secondary infection), and respective Dengue serotype ranging from picog/mL (pg/mL) to microg/ mL (μg/mL) [34,35]. While conventional immunoassay (ELISA) remains impotent in detecting NS1 within first few days of the fever onset, other attempts including Platelia™, PanBio™, Biorad, Ag-ELISA, and ultrasensitive hetero-sandwich ELISA further enhanced the NS1 detection signal and reduced the limit of detection (LOD) [27,33,34,36–42]. Platelia™ has demonstrated an average sensitivity of 83% although there is no report on the LOD level when using this technique (Table 1) [37–39]. PanBio™ suffers from insufficient sensitivity (average 69%), and the LOD of this technique was reported to be 250 ng/mL (Table 1) [36,39,40]. Biorad further enhanced the detection sensitivity (89%) of NS1 detection with no apparent LOD record (Table 1) [39]. A customized ELISA assay has described LOD of 7 ng/ mL for biorecognition of NS1 proteins (Table 1) [33]. Relying upon a low pH glycine buffer treatment, an ultrasensitive hetero-sandwich ELISA recorded LOD of ~31 pg/mL for NS1 detection while Ag-ELISA reported ~ 30 pg/mL for its LOD value (Table 1) [34,42]. To this end, no electrospun fiber-assisted platform following principles of gold standard immunoassay has reported early diagnosis of Dengue NS1 proteins with a detection outcome in pg/mL range. This study follows the gold standard protocol while prioritizing high sensitivity, stability of the material, tailored physical and chemical properties, and a considerably extended shelf life in development of the presented fiber-based devices. A combination of far field electrospinning (FFES) and a free-radical polymerization reaction was used for one-step fabrication of electrospun polymer fiber mats with desirable chemistry and physical properties. Blended polycaprolactone (PCL) electrospun fibers with different compositions of poly methyl methacrylate-co-methacrylic acid (poly(MMA-co-MAA)) offer a large specific surface area of the fibers while benefitting from the presence of surface carboxyl (-COOH) groups and eliminating the need for treatment/

detection of bacterial vaginosis and monitoring pregnancy diseases [14]. Triple-blend electrospun fiber mat was utilized for clinical diagnostic of colorectal cancer [15]. Wu et al., reported electrospun poly(εcaprolactone) (PCL) fibers for fluorescent bio-recognition of antibody against human serum albumin (anti-HSA) [16]. Polyvinylidene fluoride (PVDF) nano-fiber membrane was developed for protein immobilization using Western blotting procedure [17]. Via glutaraldhyde (GA) coupling, electrospun ammonia plasma-treated polyacrylonitryle fibers have served as bio-sensing platforms for recognition of intravenous immunoglobulin (IVIG) as a model protein [18]. In a proof-of-concept study, the high surface area of polyethersolfone (PES) electrospun fibers was used as a beneficial feature for detection of anti-staphylococcus enterotoxin B (anti-SEB). This method has relied upon carbodiimide chemistry for coupling biomolecules with the surface of the electrospun fibers [19]. With the aim of electrospinning, Chantasirichot and Ishihara developed a phospholipid polymer substrate as a biomoleculefriendly platform for conducting immunoassay. This bio-receptive electrospun surface was benefited from the inherent presence of active ester groups in its chemical structure [20]. In comparison to polystyrene (PS) substrate, which is the commercial material for fabrication of 96-well plates, phospholipid electrospun surface has shown higher specificity and greater signal to noise ratio [20]. Polyhydroxybutyrate (PHB) electrospun fibers were used as supporting substrate for fabrication of chemically functionalized bioreceptor surfaces with inherent carboxyl (-COOH) groups. By using this class of fibers, physical and covalent protein immobilization techniques were applied for Dengue virus detection and a substantially greater detection signal was obtained from developed platform when compared to conventional assay [21]. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-based electrospun fibers were integrated into a new design of 96-well plate for detection of enveloped Dengue virus. This design (i-ELISA) incorporated the electrospun mats into assay via cylinder probes attached to the lid while the body of the well plate remained intact. This platform enhanced the detection signal 12-fold while the protocol matched that of gold standard method [22]. C-reactive protein (CRP) as a cardiac biomarker was detected in the Pico-molar range by electrospun poly-Llactic acid (PLLA) fibers [23]. As another example, electrospun polystyrene–poly(styrene-co-maleic anhydride) (PS–PSMA) fibers were developed for immobilization of aptamers and subsequent application as aptasensor [24]. In the mentioned study, fluorescein dye and quantum dots have respectively shown Pico-molar and Nano-molar ranged detection capabilities when used as labels for aptamers [24]. Prevalent in tropical and subtropical countries, Dengue fever (DF) is one of the most fatal illnesses which can be contracted by travelers thus considered a worldwide threat [25,26]. Dengue initiates with a fever which has the potential to develop into more severe manifestations including Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS) [27,28]. With yearly 400 million infection cases and 21,000 deceases, early diagnosis of DF remains a challenge [29]. Major 2

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Fig. 1. Prototype fabrication: LEGO-like segments of the cylinder probes were designed and 3D printed. The complementary pieces of cylinder probes clamped electrospun fibers in between without any need for adhesive materials (a, b). The lid of the conventional 96-well plate was modified to incorporate cylinder probes and to suspend the chemically designed bio-receptive electrospun fibers inside the assay (c, d, e, and f) once the FPAP is fully developed (g and h).

functionalization processes. Electrospun fibers were thoroughly analyzed by different techniques, including scanning electron microscopy equipped with a field emission gun (FESEM), diameter range analysis, thermogravimetric analysis (TGA), water-in-air contact angle (WCA) analysis, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). These cost-effective customized fiber mats were utilized in a new design of 96-well plate namely fiber probe analytical platform (FPAP). This fiber-based microplate with an adopted integration approach from LEGO toys incorporates fiber segments through 3D-printed extensions on the lid (Fig. 1). Although prototype fabrication was done through 3D printing, at the mass manufacturing level, FPAP could be produced through an injection molding process, and hence is highly cost-effective in comparison to the competitive products. LEGO-like FPAP has shown major advantages over previous paper−/fiber-based 96-well plates including operation according to the exact clinical protocol, incorporation of highly interactive fiber mats inside the assay, high surface area available for analyte-surface interaction, and ease of application. Following an indirect ELISA protocol, the proposed platform has successfully detected NS1 proteins of Dengue virus in a considerably low quantity (0.5 pg/mL) that is promising for the early diagnosis of fatal infectious diseases, including DF. Furthermore, FPAP demonstrated an enhanced signal intensity, and improved sensitivity, specificity, and accuracy of the assay. Compared to our previous platform (i-ELISA) current platform offers inexpensive supporting substrate (PCL) and ease of electrospinning process (one step fabrication of blended fibers). Furthermore, by vertically positioning fiber mats in the assay, both sides of bioreceptors interact with the proteins hence larger specific surface area for biomolecular interaction.

This strategy also avoids any adhesive components in close proximity of biomolecules which could be a potential threat to any assay. LEGO-like FPAP holds potentials for integration of different types of papers/fibers with pre-designed specific properties and pre-immobilized proteins for the simultaneous bio-recognition of multiple target analytes. 2. Materials and methods A list of materials, chemicals, reagents, equipment, and analytical techniques with their detailed specifications are provided in the Supplementary section 1. 2.1. Synthesis reaction Four different compositions of poly(MMA-co-MAA) were prepared by free-radical polymerization reaction in THF using AIBN as an initiator. The abbreviations of the copolymers are used to identify the initial molar ratios of the monomers. For instance, poly(MMA-co-MAA9:1) corresponds to 90% of MMA and 10% of MAA in reaction mixture. Other copolymer compositions are as follows: poly(MMA-co-MAA-7:3) and poly(MMA-co-MAA-5:5). For the ease of discussion, further in the text, the mentioned copolymer compositions are referred to as Comp. (9:1), Comp. (7:3), and Comp. (5:5). Pure PMMA (when MMA was the only monomer involved in polymerization reaction) was also synthesized under the same reaction conditions and used as control in all experiments. A two-neck, round-bottom flask was fitted with a condenser and sealed inlet for reactant feed. The setup was charged with a pre-calculated amount of MMA in 50 mL of THF under stirring 3

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b). Developed fiber platforms of different classes were placed between the complimentary LEGO-like segments. The fully developed platform (Fig. 1h) introduced bio-receptive surfaces (fibers) into the assay in suspended positions.

condition for 5 min. A mixture of second monomer (MAA) and initiator (AIBN, 0.164 g) was gradually added (over a period of 1 min) to the reaction mixture. Polymerization was carried out for 8 h at 65 °C. The reaction was stopped by adding the reaction mixture into 1000 mL of distilled water. Immediate white-colored precipitations confirmed the formation of copolymer compositions. Polymer compositions were filtered and thoroughly washed with distilled water. Careful washing and filtering in this step significantly impacts the quality of the products and the yield in the synthetic reaction. Polymer compositions were dried in a vacuum oven at 40 °C for 24 h.

2.5. Application of FPAP in detection of NS1 proteins of Dengue virus 2.5.1. Buffers preparation The stock solution of PBS (10×) was prepared by adding 161.2 g of NaCl, 4.4 g of KCl, 24 g of Na2HPO4, and 4 g of KH2PO4 to 2000 mL of distilled water (DW). Diluted PBS (1×) buffer was used as a washing buffer after coating the samples with NS1 protein. Coating buffer was prepared in DW using sodium carbonate and sodium bicarbonate (2 mL of Na2CO3 (0.2 M) and 23 mL of NaHCO3 (0.2 M) in 100 mL of DW). This buffer was used for the preparation of the NS1 solution. BSA (3% in PBS 1×) was used as the blocking buffer to reduce the chance of nonspecific binding in case of non-specific binding, the blocking step was not effectively performed. Tween-20 (0.05% in PBS 1×) was employed as the washing buffer after coating samples with primary and secondary antibodies. BSA (1% in PBS 1×) was used as diluting buffer for preparation of the antibody solutions.

2.2. Electrospinning 2.2.1. Solution preparation A co-solvent of THF:DMF with the ratio 9:1, respectively, was used to prepare all solutions. All solutions were separately prepared in condensed flasks under stirring conditions at 60 °C using a hotplate for approximately 3 h until clear solutions were obtained. PMMA, poly (MMA-co-MAA) compositions, and PCL solutions were prepared as 7% w/v solutions, separately. Once fully dissolved, the two solutions were blended (PCL with PMMA and with poly(MMA-co-MAA)) with the ration of 1:1. PCL solution for the fabrication of pure PCL fibers was prepared by dissolving 2 g of PCL in 20 mL of the co-solvent solution (THF:DMF in the ratio 9:1). For blended samples (PCL in addition to PMMA or poly(MMA-co-MAA) compositions), 1.4 g of each were separately dissolved in 10 mL of the same co-solvent solution under the same conditions explained for preparation of the solutions. Blending of the two solutions was performed according to pre-calculated ratios when the heating stopped, and the solutions cooled down. Mixing of the two solutions continued overnight using a magnetic stirrer. The thorough mixing of the polymers played a vital role in uniformity of the solutions.

2.5.2. Indirect ELISA procedure and evaluation of the assay Different compositions of the electrospun fiber mats were integrated into the developed 96-well plate and an indirect ELISA procedure was carried out to detect NS1 proteins of Dengue virus serotype 2. It is important to note that the exact same procedure as described below was also performed for the fiber mats integrated into the old design of 96well plate (i-ELISA) to have a careful comparison between the performances of the two platforms. In an indirect assay, a secondary antibody is selected if only it recognizes the host species used in production of the primary antibody. A secondary antibody binds from its V region to the C region of a primary antibody while the primary antibody, in turn, is attached to the target analyte. Each well of the FPAP was charged with 150 μL of NS1 solution (concentrations: 5, 50, 5 × 102, 5 × 103, 5 × 104, and 5 × 105 pg/ mL). For each concentration, 12 positive and 8 negative replicates were conducted. Negative replicates were prepared in the absence of NS1. Incubation was carried out overnight at 4 °C (Fig. 2, step 1). Washing step was performed with 200 μL per well of PBS (1×) buffer at room temperature. FPAP well plate was washed 3 times (each time for 5 min) by using shaker with the shaking speed of 1000 rpm. Washing steps are of critical importance to avoid background signals. To promote high selectivity and to avoid non-specific binding, the blocking procedure was performed by adding 200 μL of blocking buffer to each well (Fig. 2, step 2). The incubation was carried out at 37 °C for 1 h. The choice of blocking agent might differ depending on the type of assay performed. Primary Ab solution was prepared (1:10,000) in diluting buffer. Each well has received 150 μL of primary Ab solution and was incubated for 2 h at 37 °C (Fig. 2, step 3). Washing step was performed with 200 μL per well of Tween-20 solution at room temperature. FPAP well plate was washed 3 times (each time 5 min) by using a shaker with the shaking speed of 1000 rpm. The last incubation was carried out by adding 150 μL of secondary Ab solution (1:100,000) in diluting buffer following the incubation for 1 h at 37 °C (Fig. 2, step 4). FPAP well plate was thoroughly washed as described before. Each well was charged with 100 μL of TMB ELISA substrate and incubated for 10 min (Fig. 2, step 5). Consistency in the incubation time after addition of substrate is of great importance to avoid unanticipated outcomes. Absorbance was recorded at 370 nm as the manufacture's guidelines for the biomolecules suggested. The appropriate choice of setting for readout is essential. Calculation of sensitivity and specificity was performed by using the equations below [43–45]:

2.2.2. One step fabrication of blended fibers An electric field of 12 kV (Gamma High Voltage Research, Ormond Beach, FL, USA) was used for electrospinning of all solutions. A blunt needle (0.9 mm inner diameter) was fixed at 18 cm from an aluminum collector in a horizontal position. The feeding rate was adjusted at 3 mL/h using a syringe pump. The wires from the positive and negative terminals of the capacitor were connected to the needle and aluminum collector, respectively. The whole setup (Fig. S. 1), except for the transformer, was placed inside a fume hood. The fume hood glass panel was kept closed during the electrospinning time and the fan was adjusted at a low speed to avoid deflecting the electrospinning jet. A hygrometer probe was placed inside the acrylic box to monitor relative temperature and humidity. During the electrospinning process, relative humidity and temperature were kept constant. Humidity and temperature were kept in the range of 28 ± 2 °C and 61–65%, respectively. For each set of samples, 20 mL of the solution was electrospun within 8 h. The fiber mesh collected on the aluminum foil was dried in a vacuum oven for 24 h (40 °C) to remove the residual solvent. 2.3. 3-D printing Complimentary LEGO-like segments were manufactured using a Fortus 400mc FDM system from ABS-M30 plastics. The print scheme was designed using SolidWorks software and exported to a STL file before printing (Fig. S. 2). A precise design of the LEGO segments played an important role in fabrication of the prototype. The homogeneous layer thickness was set on 120 μm. One hundred pairs of pieces were printed for development of each 96-well plate (Fig. S. 3). 2.4. Prototype development The LEGO-like cylindrical pairs with the total dimension of 5 mm were attached to the lid of the well plate using PSA layers (Fig. 1a and 4

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Fig. 2. Breakdown of the indirect ELISA protocol.

Sensitivity =

TP × 100 TP + FN

Specificity =

TN × 100 TN + FP

SEM images of the fiber mats are shown in different chemical compositions. Cross section images of the representative fibers (PCL and PCLComp. (9:1), Fig. S. 4) depict comparable morphologies for pure and blended fibers. Recorded images have shown consistent morphologies for different fiber categories, including PCL (Fig. 3a), PCL blended with PMMA (Fig. 3b), and PCL blended with poly(MMA-co-MAA) compositions (Fig. 3c–e). Formation of spindle-like beads in the structure of the electrospun fibers was observed, which are the byproducts of electrospinning. Several factors were reported to be involved in the formation of beaded fibers, including high surface tension, viscoelastic properties of the solution, and low charge density [48]. Some studies report that the applied voltage, needle to collector distance, and solution concentration are the main parameters through which the beads morphology in electrospun fibers can be controlled [49,50]. Particularly, in the case of blended polymeric solutions, such beads are typically formed due to the viscoelastic properties of a polymer solution in contrast to its stretchability [51]. In this study, we have maintained the exact same electrospinning parameters for fabrication of different PCLpoly (MMA-co-MAA) fiber compositions. The parameters were set based upon quality and integrity of the fiber materials, which were expected to sustain in tedious ELISA procedure. Supplementary Fig. S. 5 represents the diameter range analysis of the fibers with different chemical compositions. Diameter range analysis was performed by ImageJ software based on ~50 readings for each sample from three different SEM images. The beads, fiber overlapping, and crossings were avoided in the measurements. Only the fibers with clear borders were measured to reduce the chance for any errors. The average diameter was recorded between 400 nm to 600 nm, which indicates a relative consistency in fiber production (Fig. S. 5). Thermal degradation analyses were performed by TGA on pure PCL and blended electrospun fibers to identify the weight loss (W, %) of the

Variables in these equations are as follows [43–45]: True positive (TP): Positive samples that were detected as positive. True negative (TN): Negative samples that were detected as negative. False positive (FP): Negative samples that were detected as positive. False negative (FN): Positive samples that were detected as negative. The accuracy of the assay was calculated by using true negative and positive readings in comparison to the total number of replicates using the equation below [46]:

Accuracy =

TP + TN × 100 TOTAL

Limit of detection (LoD) for each separate platform was calculated from the standard deviations in the case of minimum analyte concentration (s) and slopes of the corresponding calibration plot (m) following the equation below [47]:

LoD =

3×s m

3. Results and discussion 3.1. Characterization of the fibers Morphology of the electrospun fibers was analyzed in the frontal (Fig. 3a–e) and cross-section views (Fig. S. 4) and the representative 5

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Fig. 3. Morphology analysis of the electrospun fibers by SEM: (a) PCL; (b) PCL-PMMA; (c) PCL-Comp. (9:1); (d) PCL-Comp. (7:3); and (e) PCL-Comp. (5:5).

the reported values in the literature [52,53]. In the presented plot (Fig. 4a, blue plot), the material is 100% comprised of PCL. However, for PCL-PMMA (Fig. 4a, orange plot) the onset of degradation has slightly decreased. This could be explained by the lower average molecular weight of synthesized PMMA compared to PCL resulting in a slight reduction in thermostability of the blended samples. As expected, the carbon content (residual amount at 600 °C) of the blended sample is also higher than PCL, which is in line with XPS results presented further in the article. Thorough TGA analysis for other chemical compositions are given in Supplementary section 4 (Supplementary Fig. S. 6). As can be seen, after addition of MAA to copolymers the degradation trend has changed and the values for onset of degradation and maximum decomposition rate temperature increased (Figs. S. 6. c and d). Comp. (5:5), which has the highest amount of MAA has shown the closest thermal behavior to pure PCL, which is an indication of improved

compounds and to measure the temperature for onset of degradation (TOD). This analytical technique indirectly confirms the double-phased nature of the fabricated fibers by providing separate shoulders/peaks and onsets of degradation for each individual phase. Samples were analyzed in an inert helium atmosphere (5.5 purity, SIAD TP) and the gas flow was kept constant at 30 cm3/min at a pressure of 101.325 kPa. A platinum crucible (weight ~4 mg) was used as a sample holder. The temperature in the TGA experiments was increased at a rate of 20 °C/ min starting from ambient temperature and up to 600 °C. Thermal degradation analysis of the representative fibers (Comp. (9:1)) is shown in Fig. 4a. As can be observed, the TGA plot shows a single peak for PCL, while in the case of PCL blended with PMMA the thermogram shows two individual degradation peaks, which is an indication that the material is double-phased. The onset of degradation for PCL was found to be at TOD ~ 290 °C, which is in agreement with 6

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Fig. 4. TGA analysis of the pure PCL fibers and PCL-Comp. (9:1) blended fibers (a); and XPS survey spectrum of PCL-Comp. (9:1) composite fiber with marked regions for O1s and C1s (b). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Comp. (5:5)). WCA was measured for both PCL fibers blended with poly(MMA-coMAA) composites and pure PCL (control). The results are presented in Table 2 and Supplementary Fig. S. 7. Recorded WCA values of > 90° were expected as both PCL and PMMA are considered to be moderately hydrophobic materials. Introduction of PMMA into the electrospinning mixture increased the hydrophobicity/WCA of the composite material from 114° (PCL, control) to 128° (PCL-PMMA) due to the presence of –CH3 functional groups that are hydrophobic in their nature (Fig. S. 7 (a and b)). Addition of MAA monomers to the composite fibers, however, caused a subsequent decrease in WCA from 128° to 121° due to the presence of surface –COOH groups and an overall increase in surface oxygen (the average WCA values are presented in Table 2 and the

thermostability in copolymers containing MAA. A representative survey XPS of electrospun fibers (pure PCL fibers and PCL-Comp. (9:1)) with typical C1s and O1s photoelectron signals is shown in Fig. 4b. An obvious presence of surface Si was detected in all the samples. A quantitative analysis was performed, and the results showed that Si reached a maximum value of 8.4 at.% (Table 2) in PCLComp. (7:3) samples. For that reason, C1s and O1s peak integrals showed inconsistent values for this copolymer composition in comparison to other samples. As expected, the overall C1s at. % decreased from PCL-PMMA to PCL-Comp. (5:5) as a consequence of gradual replacement of MMA with MAA units in co-polymer building blocks [54]. This gradual change in surface chemical composition was confirmed by an increase in O1s at % values from 14.65 (PCL-PMMA) to 17.39 (PCL7

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in the body in small concentrations at the onset of Dengue fever (DF) [56]. The developed electrospun fiber mats were integrated within the new and the previous design of the 96-well plate presented by our group (i-ELISA). For reminder, i-ELISA incorporated the fiber mats within the assay through cylinder-shaped pillars that modified the lid of the 96-well plate [22]. By using double-sided PSA layers, one side of the customized fiber materials was allowed to come in close contact with involving biomolecules within the assay. The new design (FPAP), however, has two major advantages over the previous design. Firstly, both sides of the bio-receptive surfaces are exposed to the bio-molecular reaction mixture. The complementary LEGO parts suspend the fiber mats within the assay in the vertical position. This allows a higher surface area (both sides of the fiber mats) to engage in analyte-surface interaction. Moreover, benefiting from the function of the LEGO segments, the new design avoids using adhesive components in close proximity to the biomolecules, which are typically a potential threat to the bioactivity of the biomolecules. It is also noteworthy that i-ELISA was previously presented with another class of fiber material which was developed in a two-step fabrication process [21,22]. Current fiber materials are the result of a one-step fabrication strategy that promotes a functional and inexpensiveness bioreceptor platform. A comparison in detection performance among the bio-receptive platforms is shown in Fig. 6. Results in this chart are presented after deduction of the cut-off values. An enhancement of 8-fold in the NS1 detection was observed in comparison to the conventional ELISA. A comparison between different compositions has shown that PCL-Comp. (9:1) offers strong detection signals within the concentration range in which ELISA was incapable of detecting the analyte of interest. Overall, the detection performance over the dynamic range has shown 4–8 fold improvement. Detection performance of the new class of fibers in the previous design has shown an overall lower performance (~2-fold) compared to the new design (Fig. 7a). This is expected as a smaller surface area is involved in analyte-surface interactions due to the one-sided design of the previous well plate. The data in Figs. 6 and 7 are presented after deduction of the cut-off values (calculated from background signal presented in inset of Figs. 6 and S. 9). Negative detection outcomes in iELISA proved PCL-Comp. (9:1) to be the reliable platform in comparison to the conventional assay and other compositions. When concentrating solely on the performance of the optimal platform in this study (PCL-Comp. (9:1)), a significant enhancement can be observed from the new design (FPAP) in contrast to the previous design (i-ELISA) as well as conventional assay (Fig. 7b). Moreover, the t-test statistical analysis was performed to evaluate the significant difference among the detection performances of conventional ELISA, i-ELISA, and FPAP. The t-test analysis shows that both platforms (i-ELISA and FPAP) present significant detection improvement in comparison to the conventional assay, with p values of 0.0023 and 0.00035, respectively. In particular, the reported p value for FPAP shows a highly statistically significant performance improvement when compared to the gold standard. The statistical analysis also shows that FPAP offers further improvement (p value of 0.0015) when compared to the previously designed platform, iELISA. The conducted assays using different fiber platforms were thoroughly calibrated and the analytical performance was evaluated. Several different concentrations of NS1 protein were targeted in an indirect ELISA assay, which is one of the most frequently applied assay protocols in clinical practices. The details of the conducted assay are presented in Fig. 2. Calibration curve analyses are presented in Fig. 8. Following the indirect ELISA protocol, NS1 was detected within pg/mL range, while the lowest concentration of NS1 that the gold standard method, ELISA, was able to detect was within the ng/mL range [57]. From a total of 72 positive replicates, conventional ELISA has detected 17 samples as negative hence imposing a considerable cut-off value over detection outcomes. This together with false negative (FN) outcomes of conventional assay has contributed to the low sensitivity and

Table 2 A breakdown of XPS and WCA data: WCA values are the average of 5 measurements (4 droplets on the corners and 1 droplet on the center of the samples) n = 3; presented WCAs are the mean values of 15 measurements ± 2°. Compositions

PCL PCL-PMMA PCL-Comp. (9:1) PCL-Comp. (7:3) PCL-Comp. (5:5)

XPS

WCA

C (%)

O (%)

Si (%)

(°)

78 83 82 75 81

16 14 16 16 17

6 2 2 8 1

114 128 126 123 121

representative images are shown in Fig. S. 7 (c and d)). It is known that –COOH functional groups are hydrophilic in their nature [55]. The trend of imposing this hydrophilic property to the surface can be clearly observed in the WCA results. In the case of Comp. (5:5), the excess of the MAA monomers in the reaction mixture resulted in an additional decrease in WCA due to the gel-like characteristic of this copolymer composition. Surface topography and surface roughness of the developed fiber platforms were analyzed by AFM. Table 3 represents selected roughness data. The spindle-like micro beads, which were observed in SEM analysis, were also detected in recorded AFM topographies (Fig. 5a and b). Fig. 5c and d depict the topography of PCL pure fibers and PCL-Comp. (9:1) fibers, respectively. Other topographies are presented in Supplementary section 6 (Fig. S. 8). Presented data in Table 3 shows an initial decrease in the roughness from PCL to PCL-PMMA, which was followed by an increase in roughness in the case of PCL-Comp. (9:1) and PCLComp. (7:3) (Table 3). Measured roughness parameters for PCL-Comp. (5:5) showed lower mean square roughness in comparison to other copolymer compositions (Table 3). Presented roughness parameters are an indirect confirmation of a systematic replacement of MMA monomers by MAA monomer in poly (MMA-co-MAA). When comparing PCLPMAA with PCL-Comp. (9:1), an increase in the mean square roughness can be observed (1087 nm to 1423 nm). Expectedly, this increase in roughness was further enhanced as the molar ratio of the MAA monomer was increased in PCL-Comp. (7:3) (1980 nm). This trend was followed by a decrease in the roughness value in the case of PCL-Comp. (5:5) (1700 nm) due to the high molar ratio of the MAA monomers (50%) in this specific composition. 3.2. Applications of LEGO-like fiber probe analytical assay The functionalized electrospun fiber mats were incorporated within FPAP for the purpose of bio-recognition. As a primary application, we have identified the need for early diagnosis of life-threatening illnesses, such as neglected tropical diseases (NTDs). We specifically concentered on early Dengue diagnosis, as this mosquito-borne viral infection is a major threat to public health. The target analyte was chosen to be nonstructural protein NS1 of Dengue virus as this biomarker circulates Table 3 A breakdown of mean square roughness and surface roughness; roughness values are the average of 3 measurements on the fiber mats of different chemical compositions (n = 3). Presented roughness parameters area are the mean values of 9 measurements ± 28 nm and ± 11 μm2, respectively.

PCL PCL-PMMA PCL-Comp. (9:1) PCL-Comp. (7:3) PCL-Comp. (5:5)

Mean square roughness (nm)

Surface area (μm2)

1963 1087 1423 1980 1700

373 508 351 169 180

8

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Fig. 5. FESEM (a) and AFM data (b, c, d): Topography analyses of the pure PCL (c) and PCL-Comp. (9:1) (d) show a distinct difference in surface roughness.

interact with cell membranes [60]. In the same fashion, from its hydrophobic regimes, NS1 can be attracted to the surface of the hydrophobic electrospun fibers (average θ ~ 122). It is also believed that NS1 has glycosidic sites with abundance of hydroxyl (-OH) functional groups [61]. The -OH chemical functional groups of NS1 can engage in hydrogen binding with the surrounding biomolecules [62]. The same force can play role in analyte-surface interaction between NS1 protein and the surface of the electrospun fibers with surface -COOH groups (Fig. 7c).

specificity for conventional ELISA in detection of NS1 (Table 4). The calibration plots for the newly developed platform (FPAP) have shown an acceptable linearity for all the analyzed samples (Fig. 8). PCL-PMMA and PCL-Comp. (9:1) have shown the highest coefficient of correlation values with R2 = 0.989 and R2 = 0.996, respectively. Among different compositions, PCL-Comp. (9:1) has shown superior performance with 100% sensitivity, 93% specificity, 97% accuracy, and a LOD of 0.5 pg/ mL (Table 4). When the same class of fiber mats was integrated within our previous design of 96-well plate (i-ELISA), we have recorded the LOD of 1.4 pg/mL. The recorded LOD from the new design (FPAP) have shown a ~3-fold improvement in comparison to integrated fiber mats within i-ELISA. Other evaluation parameters including sensitivity (97%), specificity (87%), and accuracy (94%) in the old design (iELISA) were found to be slightly lower than those of FPAP. From the chemical perspective, we anticipated that the chemicallydesigned fiber probes would attract the NS1 proteins towards the surface through dominant physical forces such as hydrogen bonding and hydrophobic interaction (Fig. 7c). The presence of surface –COOH groups could equally facilitate covalent immobilization of the biomolecules to the surface through carbodiimide chemistry or application of amine-bearing spacers (experiments were not performed in this study) [8,58,59]. In Fig. 7c, we present the potential interactions between NS1 protein and chemical functional groups available on the surface of the fibers. As it was mentioned, the dominant forces involved in analytesurface interaction are expected to be hydrogen bonding and hydrophobic interaction. Akey et al. described that NS1 possesses certain hydrophobic protrusion in its hexameric conformation that allows it to

3.3. Confirmation of the presence of NS1 on the bioreceptive surface PCL-Comp. (9:1) before and after NS1 immobilization was analyzed with Raman with an excitation source of 633 nm laser. PCL-Comp. (9:1) was chosen as the representative sample for these experiments due to its supreme performance among the rest. Raman analysis was performed with the power set at 300 mW and the exposure time at 5 s. The spectral region of 100 cm−1 to 3000 cm−1 was recorded with interval of 2.3 cm−1. The microscope objective lens was set at 50× with working distance of approximately 1.9 mm. Raman analysis was repeated (n = 3) to ensure reproducibility of spectra. For classification purpose, 10 spectra for each sample was recorded. All experiments were performed at room temperature (22 °C). The AFM analysis followed the reported methodology. Fig. 9 shows the Raman spectra of PCL-Comp. (9:1) with NS1 proteins (Fig. 9a) in contrast to the fibers without proteins (Fig. 9b). Majority of the identified peaks are in close agreement with the reported 9

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Fig. 6. Performance analysis of the pure PCL and PCL-poly(MMA-co-MAA) blended electrospun fibers in detection of NS1 proteins of DENV. For each concentration, 12 positive and 8 negative replicates were conducted. Detection results are plotted after deduction of the cut-off values (2 × average negative signal recorded for each sample). Inset plot indicates the results of the negative controls. Negative replicates were prepared in the absence of NS1.

3.4. Limitations of LEGO-like fiber probe analytical assay

spectra in the literature (Fig. 9b) [63–66]. From the observed Raman peaks, the band at 2908 cm−1 is the most prominent which is associated to the CeH stretching vibration [63–65]. The band at 1723 cm−1 is attributed to the combination of ν (C=C) and ν (C–COO) modes. The band at 1449 cm−1 is attributed to δa (CeH) of α-CH3, and δa(CeH) of O–CH3 while the assignment at 1310 cm−1 could be due to theν (CeO), ν (C–COO) vibration. Furthermore, the peak at 1058 is attributed to ν (CeC) skeletal mode, while the peaks at 853 cm−1 and 602 cm−1 are associated to ν (CH2) as well as ν (C–COO) and νs (C–C–O), respectively. Addition of NS1 to the fibers has created an overall boost in the intensity of the major peaks as marked with the symbol (*) in Fig. 9a. The enhanced peak intensity in the reports of literature is associated with the presence of NS1 proteins [67–69]. An additional contribution in the form of a shoulder in close proximity to the peak at 1449 cm−1 can be observed in the spectrum of PCL-Comp. (9:1) after NS1 immobilization (Fig. 9a, circled around). This shoulder is known to be attributed to the presence of NS1 in literature [70,71]. Moreover, a sharp contribution between 1000 cm−1 and 1100 cm−1 appeared after NS1 immobilization on the surface of PCL-Comp. (9:1). According to the literature, this outstanding peak is also associated with the presence of NS1 on the surface. This is in line with the reports of the literature [72,73]. Moreover, the AFM analysis was performed on PCL-Comp. (9:1) with NS1 proteins (Fig. 9c) in contrast to the fibers without proteins (Fig. 9d). The results demonstrate a clear alteration of the surface topography after immobilization of NS1 proteins. Recorded root mean square roughness (Rq) of the representative sample after incubation with the NS1 antigen has shown a considerable decrease from 1423 nm to 818 nm. This significant decrease in the roughness is attributed to the conglomeration of NS1 globular structures [67]. This observation in addition to those of Raman provides a clear conformation of binding of the NS1 antigens to the surface of the fibers via physical forces.

Although the current protocol offers a wide range of bio-receptive electrospun surfaces for biomolecule immobilization and subsequent detection, not all these platforms have performed to a satisfactory level in bio-recognition. While the highest detection signal was obtained from PCL-PMMA, the highest error was also observed when conducting negative controls with this specific composition. The second highest false positive results for the negative replicates were obtained from the assay conducted with pure PCL fibers. Recorded false positive signals directly affected the specificity of the outcomes, thus reducing the reliability of those particular bio-receptive platforms. PCL-poly(MMA-coMAA) compositions have shown higher performance level while the signal intensities obtained for negative replicates were within an acceptable range (Fig. 6, inset). Overall, PCL-Comp. (9:1) can be considered as the optimal bio-receptive platform offering considerable detection outcomes while having a low level of background noise. 3.5. Comparison with other methods i. FPAP was fabricated by the synergy of three techniques: free-radical polymerization reaction, electrospinning, and 3D printing. Other techniques such as printing, patterning, waxing, heating etc. are typically used in the fabrication of paper-based analytical devices [74–77]. The majority of the paper-based platforms use commercially available NC products. There is inadequate control over the NC properties, as the quality of the paper may vary from one manufacturer to another [9,74–77]. In FPAP, customized fiber materials were produced via a one-step electrospinning fabrication protocol. These fibers possess tailored chemical and physical properties. To our best of knowledge, our team is the first to have combined the two polymer systems (PCL and poly(MMA-co-MAA)) 10

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Fig. 7. Performance analysis of the pure PCL and PCL-poly(MMA-co-MAA) blended electrospun fibers in detection of NS1 protein of DENV integrated in our previous well plate design, intrant ELISA (I-ELISA) (a). For each concentration, 12 positive and 8 negative replicates were conducted. Detection results are plotted after deduction of the cut-off values (2 × average negative signal recorded for each sample, please see the supplementary information, Fig. S. 15). A comparison between the performances of three platforms, FPAP, i-ELISA, and conventional ELISA, in detection of NS1 protein (b). The significant differences between i-ELISA and FPAP are compared with conventional ELISA by t-Test. The probability (p) values < 0.05 are considered to be statistically significant. Presented results were recorded from Comp. (9:1) as the representative bio-receptive platform integrated within different designs. Conventional ELISA was performed without any electrospun fiber mats. Interactions between NS1 protein of Dengue virus serotype 2 and chemical functional groups available on the surface of the fibers (c). As presented, the dominant forces involved in analyte-surface interaction are hydrogen bonding and hydrophobic interaction.

iii. NC contains –OH functional groups in its chemical structure that, in turn, require modification strategies to convert –OH groups to more reactive functional groups, such as –COOH groups [79–82]. While functionalization of the paper materials may result in generation of the desirable surface functional groups, the stability of the paper is typically affected by such treatments [83–88]. Developed fiber materials in this study benefit from the inherent presence of surface –COOH groups in an optimized concentration, thus eliminating the need for further modification/functionalization of the material which may compromise its integrity.

to have produced electrospun fiber mats as the bio-receptive platforms for detection purposes. ii. Commercial NC is known to have WCA of 21° to 67° [78]. This level of hydrophilicity is not always in favor of the assay, as it may induce an increased chance of false positive signals due to the protein entrapment and incomplete washing process. For that reason, paper-based analytical platforms generally suffer from insufficient sensitivity [9]. Wettability in our developed fiber platforms can be tuned by variation of the monomer ratios in the synthesis and in the choice of supporting substrate. 11

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Fig. 8. Calibration plots for NS1 detection by developed electrospun fibers in a wide concentration range: the integrated fiber mats in FPAP (a) in contrast to the integrated fiber mats in i-ELISA (b).

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Table 4 Evaluation parameters of the assay, including sensitivity, specificity, accuracy, and limit of detection. NS1 status

Positive (TP, FP) Negative (FN, TN) Total Sensitivity (%) Specificity (%) Accuracy (%) LoD (pg/mL)

PCL

PCL-PMMA

PCL-Comp. (9:1)

–

72

20

70

19

72

3

72

5

71

0

28

2

29

0

45

0

43

1

48

72

48

72

48

72

48

72

+

–

PCL-Comp. (5:5)

+

72

–

PCL-Comp. (7:3)

+

–

+

ELISA

–

+

+

–

8

55

18

40

17

30

48

72

48

100

97

100

100

98

76

58

60

93

89

83

62

83 3.5

82 3

97 0.5

95 7.2

92 4.6

70 500

temperature [84,85,87]. Despite such efforts, the shelf life of the paper-based platforms varies from few hours to few days with the performance of the activated paper platform decreasing with time [83–88]. Using PCL as the supporting material with the life time of over 1 year combined with plastic materials (PMMA and poly (MMA-co-MAA)) that have considerably prolonged shelf life benefited FPAP with an extended shelf life of ~2 years [90]. A thorough testing of FPAP showed that the platform stored in room

iv. One of the major challenges when dealing with paper materials is the limited shelf life that is often as brief as 24 h after activation of the surface [8,54,89]. Majority of the paper-based devices which use commercial filter paper materials report strategies to extend the lifetime of these devices [83–88]. The storage conditions play a vital role in preservation of the paper platforms. Lower temperatures (−4 °C (refrigerator) and −20 °C (freezer)) were reported to promote better stability of the platforms over time than room

Fig. 9. Raman spectra of PCL-Comp. (9:1) with NS1 proteins (a) in contrast to the fibers without proteins (b). AFM analyses of PCL-Comp. (9:1) with NS1 proteins (c) in contrast to the fibers without proteins (d). The roughness bar indicates 2.04 μm. 13

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Fig. 10. Schematic comparison between a representative paper-based 96-well plate and FPAP. Red and black arrows in this scheme depict disadvantages of a representative marketed platform (a) and advantages of FPAP (b), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

fabrication was done through 3D printing, at the mass manufacturing level, FPAP could be produced through an injection molding process, and hence is highly cost-effective in comparison to the competitive products. vii. Due to its LEGO-like design, this platform offers multi-functionality (Supplementary Fig. S. 10). FPAP could host a wide range of fibers and/or papers with their specific properties and functionalities and pre-immobilized biomolecules for the simultaneous detection of several infectious agents (shown in different colors of fiber mats in Fig. 10b). viii. Compared to other existing techniques for detection of Dengue NS1 protein, FPAP has proven to be highly sensitive (100%), specific (93%), and accurate (97%) while offering the lowest reported LOD for NS1 detection (0.5 pg/mL). A thorough comparison among detection performances of this platform and other existing techniques are presented in Table 1. ix. Electrospun fiber sheets presented in this study could be potential

temperature within a vacuum desiccator preserved its integrity and tailored chemical and physical properties over time and the detection performance was comparable to that of immediately fabricated. v. FPAP allows for visual judgment of the results, which could potentially be quantified using tablets, smart phones, and/or digital desktop scanners [91]. The majority of the existing, paper-based 96-well plates incorporate paper segments at the bottom of wells or microzones, thus leaving no chance for the routine quantification of the detection results (a representative platform is shown in Fig. 10a). In FPAP, a regular signal measurement can be carried out by intensity recording of the resultant colors using an ELISA reader (Fig. 10b). vi. Unlike multi-layered complex paper-based well plates, PFAP is a two-part platform in which conducting the assay has no major differences than that of routine clinical assay (Fig. 10b). Therefore, it is convenient to be used by lab technicians. Although prototype 14

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candidates for fabrication of μPADs.

researchers through the experimental, while M. J. M. has provided the groups with the scientific insights regarding the fundamentals and applications of the new strategy. S. H. wrote the paper. All the authors have read and commented on the manuscript.

4. Conclusions Presented study in this article describes conceptualization and fabrication of fiber probe analytical platform (FPAP), which was inspired by design and function of LEGO toys. This class of fiber mat-based 96well plate allows chemically designed electrospun fibers to suspend within the assay therefore offering higher chance of analyte-surface interaction. The attachment of the NS1 proteins to the surface of the electrospun fiber mats was confirmed by Raman and AFM analyses of the samples with and without NS1 proteins. FPAP has shown effective and highly sensitive/selective detection of NS1 Dengue protein compared to established methods while offering LOD within femtog/mL range. This fiber mat-based analytical platform consists of two segments (body and lid) that operate in the exact fashion as conventional ELISA hence easy to apply in typical clinical setups. Customized fibers in this study were fabricated via one-step electrospinning of a blended copolymeric material that is benefited from inherent presence of –COOH functional groups. Moreover, FPAP offers integration of several paper/ fiber types with their specific chemical and physical characteristics within its design that is promising for simultaneous bio-recognition of biological elements.

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CRediT authorship contribution statement Samira Hosseini: Conceptualization, Data curation, Formal analysis, Supervision, Methodology, Writing - original draft. Pedram Azari: Visualization, Methodology, Validation. Braulio CardenasBenitez: Conceptualization, Methodology, Validation. Eduardo Martínez-Guerra: Formal analysis. Francisco S. Aguirre-Tostado: Formal analysis. Patricia Vázquez-Villegas: Formal analysis. Belinda Pingguan-Murphy: Resources, Funding acquisition. Marc J. Madou: Supervision. Sergio O. Martinez-Chapa: Resources, Funding acquisition. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors would like to acknowledge the financial support of Tecnologico de Monterrey, Mexico, for the special grant (grant number: 002EICII01) awarded to the Nano Sensors and Devices Focus Group, School of Engineering and Sciences, Tecnologico de Monterrey, Monterrey, Mexico. The present study was also supported by Frontier research grant (FG021-17AFR) under the Ministry of Higher Education (MOHE), Malaysia. The authors deeply appreciate the assistance of Professor Emeritus Geoffrey A. Cordell for the English editing of this manuscript. Authors would like to thank Prof. Ivan Djordjevic for his assistance in data interpretation. Authors would also like to acknowledge the technical support of the Writing Lab, TecLabs, Tecnologico de Monterrey, Mexico, in production of this work. Authors contribution S. H. synthesized the copolymer compositions. P. A. fabricated the electrospun fibers. E. M-G. and F. S. A-T. performed characterization on the fiber materials. B. C-B. conceived and designed the LEGO-like complimentary segments, while S. H. 3D printed and fabricated the prototype. S. H. and P. V-V. conducted the immunoassay for NS1 detection. S. H and P. A have conducted the immunoassay for the detection of enveloped Dengue virus. B. P-M. and S. O. M. C. have guided the 15

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[23]

[24]

[25]

[26]

[27]

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45] [46]

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