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Apr . 19, 2024 14:36 Back to list

Demonstrated via hCG Assay for Pregnancy Test

Abstract

Over the past decades, paper-based lateral flow immunoassays (LFIAs) have been extensively developed for rapid, facile, and low-cost detection of a wide array of target analytes in a point-of-care manner. Conventional home pregnancy tests are the most significant example of LFAs, which detect elevated concentrations of human chorionic gonadotrophin (hCG) in body fluids to identify early pregnancy. In this work, we have upgraded these platforms to a higher version by developing a customized microfluidic paper-based analytical device (μPAD), as the new generation of paper-based point-of-care platforms, for colorimetric immunosensing. This will offer a cost-efficient and environmentally friendly alternative platform for paper-based immunosensing, eliminating the need for nitrocellulose (NC) membrane as the substrate material. The performance of the developed platform is demonstrated by detection of hCG (as a model case) in urine samples and subsequently indicating positive or negative pregnancy. A dual-functional silane-based composite was used to treat filter paper in order to enhance the colorimetric signal intensity in the detection zones of μPADs. In addition, microfluidic pathways were designed in a manner to provide the desired regulated fluid flow, generating sufficient incubation time (delays) at the designated detection zones, and consequently enhancing the obtained signal intensity. The presented approaches allow to overcome the existing limitations of μPADs in immunosensing and will broaden their applicability to a wider range of assays. Although, the application of the developed hCG μPAD assay is mainly in qualitative (i.e., positive or negative) detection of pregnancy, the semi-quantitative measurement of hCG was also investigated, indicating the viability of this assay for sensitive detection of the target hCG analyte within the related physiological range (i.e., 10–500 ng/mL) with a LOD value down to 10 ng/mL.
Keywords: 
microfluidic paper-based analytical devices; point-of-care testing; human chorionic gonadotrophin; colorimetric detection; immunosensing; pregnancy test
 

1. Introduction

Traditional lateral flow (immuno) assays (LFIAs) are immunochromatographic paper-based platforms comprising a nitrocellulose (NC) membrane as a reaction surface with a high protein-binding capability, suitable for irreversible antibody immobilization. The NC membrane is impregnated with the corresponding capture antibodies at the specific zones, indicating the test-line and control-line for the qualitative or semi-quantitative detection of specific target analytes [1,2]. For example, conventional home pregnancy test LFAs are the most popular and widely commercialized tools for rapid, simple, and qualitative detection of elevated concentrations of human chorionic gonadotrophin (hCG) in urine or serum samples, identifying pregnancy at early stages. hCG is a glycoprotein hormone normally generated by the placenta during pregnancy. hCG molecule is composed of 237 amino acids with a molecular mass of 36.7 kDa and two subunits, the alpha and beta. Secretion of hCG starts almost a week after fertilization, which reaches to its peak (1000 ng/mL) in 8 weeks of pregnancy. After this period, the hCG level begins to decline until it stabilizes. The normal serum hCG level is less than 10 ng/mL; the detection of hCG at this level is thus significant for early pregnancy identification. Although there are various methods available for detection of hCG (e.g., ELISA), the conventional LFA based tests offer numerous advantages, such as being rapid, simple, low cost, and easy to operate [3].
Due to the natural strong affinity for proteins, the NC membrane has been traditionally used as the most favorable reaction surface for LFAs. However, there are some issues and limitations associated with the NC membrane such as the high cost, fragility, flammability, toxicity (alteration to habitats), and hydrophobicity (requires deposition of surfactants). In the past decades, there have been extensive efforts made in the LFA industries in order to replace the NC membrane with other possible alternative materials, including nylon, polyvinylidene fluoride (PVDF), polyethersulfone, polyethylene, fused silica, and other composite membranes. However, each of these materials has exhibited some challenges and drawbacks, thereby maintaining the superiority of the conventional NC membrane [4,5]. These materials are relatively costly, incompatible with the chemistry of performed bioassays, and also present a lower signal-to-noise ratio compared to NC membrane. Nevertheless, a fluorescent-based LFA was recently reported for detection of cardiac troponin I, where the filter paper was modified with carbon nanofibers and used instead of NC membrane [6].
In 2007, the Whitesides group expanded the concept of paper-based analytical devices and introduced microfluidic paper-based analytical devices (μPADs) as a new generation of paper-based point-of-care (POC) platforms [7]. They demonstrated that filter paper can be used as a viable platform for development of disposable analytical devices, presenting numerous advantages over the conventional materials utilized in the fabrication of such devices, particularly NC membranes [8]. Filter paper is a low-cost, accessible, hydrophilic, flexible, and disposable material, which makes it an ideal platform for POC (bio)chemical diagnostic testing. The emerging μPADs use patterned filter paper as a platform for POC testing, providing a great deal of flexibility for qualitative, semi-quantitative, or quantitative measurements of a wide variety of target analytes in diverse sample matrices [9,10]. We also developed a μPAD for detection of human serum albumin based on smart-phone signal readout [11]. Despite of all these benefits as well as the significant progress made in the realm of μPADs, the lack of enough protein binding capacity as one of the main limitations of the filter paper has narrowed down the application of μPADs to only some simple (bio)chemical assays where there is no need for immobilized antibodies or proteins typically required in traditional sandwich LFAs (e.g., hCG pregnancy test) [12]. Essentially, this is due to the inert nature, hydrophilicity, and neutral surface chemistry of filter paper fibers, not providing the required attraction forces to retain the antibodies, whereas the existing hydrophobic and electrostatic interaction forces allow retention of biomolecules in NC membranes. There have been some strategies reported to address this limitation, presenting different types of chemical or physical paper modification techniques to retain various (bio)chemical reagents upon paper [13,14,15,16,17,18,19,20] and also various signal amplification strategies [21,22] on paper. However, the presented methods are not usually practical enough to selectively immobilize the capture antibodies in the designated detection zones upon the surface of filter paper, enabling development of sequential multistep sandwich immunoassays. Majority of the introduced paper treatment methods are not efficient enough for selective modification of the desired detection spots upon paper by many tokens, such as involving the entire paper surface area in modification, which is not favorrable and can often cause non-specific binding and affecting the overall assay performance. In addition, these methods usually involve some tedious modification steps requiring multiple times exposure of paper to the treatment solutions, which consequently affects the 3D network of paper and deteriorates the natural capillary-based fluid flow along the paper channels [13,14,15,16,17,18,19]. For instance, Zhu et al. modified paper via oxidation of cellulose fibers using NaIO4 solution; the entire μPAD was immersed and left in this solution for about 40 min to generate aldehyde groups on the surface of cellulose. Afterwards, μPAD was washed thoroughly with water and then dried for future use [18]. In another work, various chemistries were investigated for chemical modification of filter paper for immobilization of biomolecules. Among all, modification with KIO4 was found to be the most efficient method; however, it involved incubation of the whole filter paper in aqueous solution of KIO4 at 65 °C for 2 h [23]. After, the modified paper was washed three times with water and then blotted and dried overnight. Finally, the modified paper was used for fabrication of μPADs.
In the present work, a customized μPAD was proposed as an alternative to the conventional LFA-based colorimetric immunosensing systems. The performance of the developed μPAD assay was demonstrated via qualitative and semi-quantitative detection of hCG protein in urine samples, indicating positive or negative pregnancy. This was achieved through selective immobilization of the desired capture antibodies upon the detection zones, where filter paper was impregnated with a dual-functional carboxylated silane composite, allowing formation of the sandwiched colorimetric complexes. The design, dimension, and geometry of the platform has been also optimized generating a regulated fluid flow to enhance the intensity of the obtained colorimetric signal. Both presented optimizations in surface chemistry of filter paper and also the device design, enabled detection of the target hCG protein in the tested samples and subsequently corelating them with either a positive or negative pregnancy. In addition, the presented optimized filter paper modification methods along with the new functional design of the device are superior to the previously reported works, which can be used in the future for similar applications and analytes. This will broaden the application of μPADs to a wider range of assays while eliminating the need for the NC membrane as the dominant material used for paper-based immunoassays.

2. Experimental Section

2.1. Chemicals and Materials

All chemicals were of analytical reagent grade. Human chorionic gonadotropin (hCG) protein, goat anti-alpha hCG monoclonal capture antibody, and goat anti-mouse IgG antibody, were all supplied from Sapphire Bioscience Pty. Ltd. (Redfern, NSW, Australia). Mouse anti-beta hCG monoclonal antibody-colloid gold (AuNPs) conjugate was purchased from Abcam (Melbourne, Australia). Phosphate buffer saline (PBS) (0.01 M, pH 7.4), sucrose, tween-20, tris, and bovine serum albumin (BSA), were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia). Water was treated with a Millipore (Bedford, MA, USA) Milli-Q water purification system and was used throughout. Whatman grade 4 qualitative filter paper with a pore size of 25 µm and thickness of 210 µm (GE Healthcare Australia Pty. Ltd., Parramatta, NSW, Australia) was used for fabrication of µPADs. Herein, the relatively large paper pore size (i.e., 25 µm) facilitates full transport of AuNPs probe through paper channels. Glass fiber membrane (SB06), absorbent pad, and backing pad were purchased from Shanghai Kinbio Tech. Silica gel particles (40–63 µm) were obtained from Silicycle (Quebec, QC, Canada). Silane-Polyethylene glycol (PEG)-carboxylic acid (COOH) was purchased from Nanocs Inc. (New York, NY, USA). The Freedom pregnancy test kit (Chemist Warehouse, Sydney, NSW, Australia) was used to test the real urine samples. Human chorionic gonadotropin (hCG) protein was purchased from Nanjing Santa Scott biotechnology, 0.01 M pH 7.4 PBS, Diagnostic kit for human chorionic gonadotropin (colloidal gold immunochromatography) was purchased from Beijing Zizhu pharmaceutical.

2.2. Instrumentation

Silhouette Studio® (V4.1.156) free software was used to design the microfluidic layouts. A wax printer (Colorqube 8870, Xerox, Norwalk, CT, USA) was utilized to print the microfluidic patterns upon the filter paper. A regular desktop Epson scanner was used to acquire images of µPADs. The quantitative image analysis was carried out using free image j software. A revolving handheld puncher was used to cut out circles on the paper. A scanning electron microscopy (SEM, JEOL InTouchScope, JSM-IT 500 HR, Frenchs Forest, NSW, Australia) coupled to an energy-dispersive X-ray spectroscopy (EDS, JEOL, Ex-74600U4L2Q, Frenchs Forest, NSW, Australia) were used for investigation of morphology and elemental analysis of paper device.

2.3. Design and Fabrication of µPADs

2.3.1. Wax Printing

The microfluidic patterns were designed using the Silhouette Studio® and wax printed on the paper as reported previously [24,25]. The pattern was composed of a sample zone, test zone (T), control zone (C), and a waste zone. The outline and dimensions (before and after wax melting) of the µPADs are illustrated in Figure 1a. The printed paper was then placed on a hot plate (2 min/130 °C) to melt the wax through the thickness of the filter paper. The fabrication process was continued by pipette drop-casting of the corresponding reagents upon the desired zones.
Biosensors 11 00474 g001 550
Figure 1. Fabrication process of µPADs. (a) Computer layout comprising detection, sample, and waste zones. (b) Printed pattern on the paper. (c) Melted wax. (d) Sample zone punched out and absorbent pad stacked on the waste zone. (e) Probe disk is stock on the sample zone.

2.3.2. Reagent Deposition

First, the T and C zones were treated with the silane composite (2 µL, suspension was shaken before pipetting) and then incubated in an oven (37 °C/30 min). After, the required antibodies were deposited into the T and C zones (anti-alpha hCG and anti-mouse IgG respectively, 1.5 µL of 1 mg/mL in each zone) and then incubated further in the oven (37 °C/1 h). Finally, the blocking buffer (PBS pH = 7.4 containing BSA 1% w/v, Tween-20 0.25% v/v, sucrose 2%) was loaded (total 5 µL in 1 µL increments) over the entire area of μPADs (excluding the waste zone) and incubated for another 1 h (37 °C). The number of exploited reagents was optimized in order to find out the least amount required while generating the highest assay sensitivity.

2.3.3. Final Assembly

After all the paper treatment steps, the μPADs final assembly was accomplished by punching out the sample zone area using a handheld puncher, creating a hole (diameter 5 mm) to affix the probe disk to the μPADs. Then, paper was attached to the sticky side of a backing pad providing further mechanical support. In the next step, a rectangular shaped layer of absorbent pad (10 × 20 mm) was stacked over the waste zone in order to generate extra wicking capacity, allowing larger volumes of samples being tested. Then, a probe disk was attached to the sample zone area. To prepare the probe disk, a circular disk (6 mm) was punched out from the glass membrane and then it was treated initially with the blocking buffer (5 µL) followed by addition of Anti-hCG-gold conjugate (5 µL) while being incubated in the oven (37 °C/30 min) for each treatment. The disk size (6 mm) was slightly larger than the hole (5 mm) in order to make sure that the entire area in the sample zone is covered by the disk. Prepared µPADs were kept in the fridge (4 °C) for future use. Photographs of µPADs throughout different preparation steps are shown in Figure 1.

2.4. Preparation of Silane Composite

100 mg silica gel particles (60 µm) and 3 mg silane−PEG−COOH were dispersed in 1 mL of ethanol (50% v/v) and incubated for 24 h (60 °C). Then the suspension was centrifuged and washed three times with ethanol (50% v/v). Afterward, the precipitate was redispersed in 500 µL PBS and then 200 µL of a 1:1 solution of EDC/NHS (10 mg/mL in PBS) was added and incubated further for 2 h in room temperature. Finally, the suspension was centrifuged and washed with PBS. The precipitate was eventually resuspended in 500 µL PBS and kept in dark for further use (always used in the same day).

2.5. Analysis Procedure

The hCG standard solutions were prepared by diluting a stock solution (100 μg/mL) of hCG in buffer solution (PBS with BSA 0.1% w/v). The testing was performed by loading 50 μL of hCG solution at different concentration levels (0–500 ng/mL) into the sample zone of µPADs. For the qualitative measurements, formation of a visible red color pattern at the T zone indicated a positive signal, while absence of the color was associated with the negative signal. The semi-quantitative signal readout was carried out after 20 min (stabilized color intensity) by scanning the µPADs using a desktop scanner followed by image analysis using ImageJ software. Further details in this regard are provided in the following sections.

3. Results and Discussion

3.1. Working Principle of the hCG µPAD Assay

Similar to LFA-based pregnancy tests, the presented µPAD assay is also a sandwich immunoassay, implementing AuNPs-antibody (i.e., anti-beta hCG monoclonal antibody) conjugates as the colorimetric signaling probe. As shown in Figure 2A, after loading the sample (50 μL), including hCG target protein into the sample zone, the deposited probe will be detached from the glass membrane substate and the hCG-probe complex will be formed, which will then move toward the T zone via the capillary effect. At the T zone, the complex will be captured by the immobilized hCG alpha antibody while the free probes will move even further, where they are captured by the IgG antibody present at the C zone. The appearance of two red circular pattern indicates a positive response, whereas if no hCG analyte is present in the introduced sample (i.e., no complex formation), there will not be any colored T zone, indicating a negative response.
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