title: Paper-based Point-of-Care Technology for Biomarker Detection
Creators: name: Kasetsirikul, Surasak
Publisher: name: Griffith University
Year: 2022
Subjects: subject: paper-based devices
subject: literature review
subject: liquid absorption
subject: fabrication
subject: biochemical
subject: technologies

description: Although advanced medical technology has been progressively developed, access to quality healthcare services is still a major problem, especially in developing countries. Advanced medical technology requires enormous and expensive resources; therefore, affordable, easy to use and accessible technology could bridge the gap, improving people’s lives, and attracting venture capital investment. Moreover, it is expected that the diagnostic device market would grow exponentially, particularly for paper-based technology. As a result, technology development could promote the local economy by creating jobs. Paper-based analytical devices have been introduced and developed over the last decades. This technology has been widely used as a tool for diagnosis in fields such as environment, food quality and healthcare. Point-of-care (POC) diagnosis has attracted a great deal of attention from the research community, eventually aiming for the development of a platform that can evaluate biological markers in body fluids such as saliva and urine. Paper-based diagnostic devices make an impact because of their low cost, environmentally friendliness and biodegradability. The significant advantage of paperbased devices is the capillarity driven fluid transport through the paper network without the need for additional equipment. This thesis starts with a detailed literature review on paper-based devices. The literature review includes material selection, fundamentals, applications and design criteria, and mainly discusses the technical challenges in engineering and biochemical aspects. Additionally, this section will discuss the current research trends and perspectives of advanced technologies for enhancing assay performance. The study of wicking in paper strips predicts the flow behaviour to control fluid flow and perform programmable fluid handling tasks. An accurate model to predict flow in paper is required for designing paper-based devices. In this thesis, a novel model explains wicking in paper strips altogether with liquid absorption capacity. Based on observation, paper can store liquid in a matrix because after removing the reservoir, the fluid continues flowing. We employ the electrical circuit analogy to formulate the model. The capacitance should be included in the model because the capability to store and release charges is analogous to liquid absorption capacity in the matrix which contain and discharge the fluid. The theoretical data from the model agrees well with the experimental data obtained for wicking in paper strips in a vertical configuration. Additionally, fitting the model with experimental data confirms the critical parameters of liquid absorption capacity and capillary pressure. Considering liquid absorption capacity as a capacitance in electrical circuit analogy could elucidate the relationship between materials and wicking mechanism. Next, the thesis focuses on developing of paper-based analytical device fabrication technology that allows the defined hydrophobic pattern on paper to guide fluid along a hydrophilic path in a controlled manner. There have been many fabrication processes reported in recent decades. However, some methods using harsh chemicals result in contamination of the subsequent reagents for analytical assays. Some other techniques have complicated processes requiring expensive equipment, impractical for mass production. The subsequent study focused on a parametric fabrication of parafilm hot pressing, which is inexpensive, rapid, and straightforward. The basic concept is providing heat and pressure to melt and squeeze parafilm into the paper matrix resulting in a hydrophobic pattern defined by a laminate mask. The smallest hydrophobic barrier made by this technique is 821 um, resulting from the resolution of the laminate mask. Likewise, this study also demonstrated the suitability of paper for both physical and biochemical functions. In terms of physical function, the wicking speed on fabricated paper is slower than on non-fabricated paper because the pore could be reduced due to pressure. Diffusive mixing in 2D and 3D paperfluidics are also reported. We employed a sandwich immunological assay for biochemical functions to evaluate protein binding capacity on the paper. Demonstrating the paper device from this fabrication process is potentially applicable to analytical instrument for wicking studies and biomolecule detection. Besides investigating wicking in paper strips and the fabrication process to handle engineering challenges, biomarker detection has been studied to demonstrate diagnostic applications for the developed devices. Biomarkers used in this study include SARSCoV-2 humanised antibody and cell-derived exosomes. The readout methods implemented in this study are colourimetric, fluorescent, and electrochemical. First, we employed a paper-based colorimetric assay using the horseradish peroxidase and 3,3’,5,5’-tetramethylbenzidine (TMB)/hydrogen peroxide system. The colourimetric readout was obtained from a self-made image acquisition system and is quantified using the MATLAB program. The detection limit of SARS-CoV-2 humanised antibody assay was 9.00 ng/uL, which is lower than commercially available kits (0.112 IU/mL vs 5 IU/mL). However, the result for exosome detection encountered many challenges. Firstly, the exosome concentration may be inadequate to reach a detectable range. Secondly, high background signal resulting from non-specific binding on the platform leads to a lack of sensitivity and specificity for exosome detection. A paper-based colourimetric assay has the potential to be further developed into a point-of-care diagnostic device. Further modification of the paper may be required to promote protein binding for specific targets and prevent non-specific binding to reduce the background signal. Next, the thesis reports a paper-based immunofluorescent assay for biomarker detection using fluorophore conjugation with detecting antibodies. The fluorescent-based assay requires a specific excitation wavelength and retrieves a wavelength of emission. Fluorescent microscopy was used to observe the readout. Before, the images were processed and quantified using a MATLAB program. The assay selectively detects SARS-CoV-2 humanised antibodies spiked in PBS and healthy human serum samples. The limit of detection of the assay was 2 ng/uL (0.025 IU/mL) and 10 ng/uL (0.125 IU/mL) in PBS and human serums, respectively. This assay can detect 1010 exosome/mL obtained from cell culture media, but also faced many obstacles. First, exosome concentration prepared from cell culture media may be insufficient to reach the detectable range. Second, minimising chemical contamination could enhance assay specificity and sensitivity to prevent non-specific absorption. Therefore, a paper-based fluorescent assay could be further developed into a portable device. The light source to excite the fluorophore and to emit the signal, including an optical system, could be scaled down from fluorescent microscopy into a handheld-size device. Lastly, this thesis presents a proof-of-concept electrochemical paper-based device for biomarker detection. The paper-based device is fabricated using parafilm hot pressing, as reported previously. The electrochemical chacracterisation on paper-based carbon electrodes was thoroughly investigated using cyclic voltammetry. The detection employed a sandwich immunological assay using carbon electrodes on paper. Differential pulse voltammetry was used to observe the current response in the subsequent steps. The stepwise addition of biomolecules on paper-based carbon electrodes results in the attenuation of the current response caused by stepwise biomolecules binding on the electrode surface. The current reaction after target binding corresponds to the target concentrations. In addition, electrochemical impedance spectroscopy is utilised to affirm the validity of the assay by observing the electron transfer resistance coming from the interfacial electron transfer at the electrode surface. The assay for SARS-CoV-2 antibody detection in PBS samples detected the target concentration in the range of 10 to 100 ng/uL. The detection limit is estimated to be 9.37 ng/uL. For exosome detection prepared from cell culture media, this assay quantified the total exosome and ovarian cell-derived exosome concentration with a limit of detection of 9.3 X 10 exosomes/mL and 7.1 x 10 exosomes/mL with &lt; 10% relative standard deviation for samples of n =3. However, the limit of detection can be enhanced by strengthening antibody immobilisation on the paper-based device and stabilising the carbon electrodes on paper to be conductive enough to sense the change of the subsequent loading of biomolecules. Our electrochemical paper-based assay could be an alternative tool for detecting diseasespecific exosomes in biological samples for point-of-care diagnosis. In conclusion, this study aims to overcome engineering and biochemical challenges posed by paper-based analytical devices. To tackle the engineering issues, a novel wicking model with consideration of liquid absorption capacity offers an alternative way to predict flow behaviour in capillary rise experiments and explain the material characteristics. Additionally, the thesis studies the fabrication parameters to control the paper-based device fabrication better using parafilm hot pressing, demonstrating the functionality of paper for both physical and biochemical applications. Regarding biochemical challenges, the thesis reports colourimetric, fluorescent, and electrochemical techniques implemented on paper-based platforms, employing a sandwich immunological assay to detect biomarkers, which include SARS-CoV-2 humanised antibody and cell-derived exosome samples. These established protocols have the potential to be further improved for automation and portability, which could be compatible with other advanced technologies such as wearable sensing devices, artificial intelligence and machine learning.