Paper-based Point-of-Care Technology for Biomarker Detection

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 < 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.Thesis (PhD Doctorate)Doctor of Philosophy (PhD)School of Eng & Built EnvScience, Environment, Engineering and TechnologyFull Tex

Formation of cell spheroids using Standing Surface Acoustic Wave (SSAW)

3D bioprinting becomes one of the popular approaches in the tissue engineering. In this emerging application, bioink is crucial for fabrication and functionality of constructed tissue. The use of cell spheroids as bioink can enhance the cell-cell interaction and subsequently the growth and differentiation of cells in the 3D printed construct with the minimal amount of other biomaterials. However, the conventional methods of preparing the cell spheroids have several limitations, such as long culture time, low-throughput, and medium modification. In this study, the formation of cell spheroids by SSAW was evaluated both numerically and experimentally in order to overcome the aforementioned limitations. The effects of excitation frequencies on the cell accumulation time, diameter of formed cell spheroids, and subsequently, the growth and viability of cell spheroids in the culture media over time were studied. Using the high-frequency (24.9 MHz) excitation, cell accumulation time to the pressure nodes could be reduced in comparison to that of the low-frequency (10.4 MHz) excitation, but in a smaller spheroid size. SSAW excitation at both frequencies does not affect the cell viabilities up to 7 days, > 90% with no statistical difference compared with the control group. In summary, SSAW can effectively prepare the cell spheroids as bioink for the future 3D bioprinting and various biotechnology applications (e.g., pharmaceutical drug screening and tissue engineering).ASTAR (Agency for Sci., Tech. and Research, S’pore)MOE (Min. of Education, S’pore)Published versio

Wicking in Paper Strips under Consideration of Liquid Absorption Capacity

Paper-based microfluidic devices have the potential of being a low-cost platform for diagnostic devices. Electrical circuit analogy (ECA) model has been used to model the wicking process in paper-based microfluidic devices. However, material characteristics such as absorption capacity cannot be included in the previous ECA models. This paper proposes a new model to describe the wicking process with liquid absorption in a paper strip. We observed that the fluid continues to flow in a paper strip, even after the fluid reservoir has been removed. This phenomenon is caused by the ability of the paper to store liquid in its matrix. The model presented in this paper is derived from the analogy to the current response of an electric circuit with a capacitance. All coefficients in the model are fitted with data of capillary rise experiments and compared with direct measurement of the absorption capacity. The theoretical data of the model agrees well with experimental data and the conventional Washburn model. Considering liquid absorption capacity as a capacitance helps to explain the relationship between material characteristics and the wicking mechanism.</jats:p

Challenges and perspectives in the development of paper-based lateral flow assays

Lateral flow assays (LFAs) have been introduced and developed over the last half century. This technology is widely used as a tool for diagnosis in several fields such as environment, food quality and healthcare. Point-of-care (POC) diagnosis using LFAs has been attracting attention of the research community, particularly aiming for the development of a platform that can evaluate of biological markers in bodily fluids such as saliva and urine. The existence of a disease or the pregnancy can be determined by a test device, before further investigation and medical treatment. LFAs make use of a disposable test strip, which can provide diagnosis result on the spot within minutes. Thus, LFAs is a promising alternative of preliminary diagnosis for laboratory instruments that are costly, time consuming and require trained personnel. This paper includes a brief overview of the conventional LFAs: material selection based on its roles and characteristics, working principles, fundamentals, applications, and design criteria. We mainly discuss the technical challenges in both engineering and biochemical aspects and recommends possible solutions. We identify current research trends and provide perspectives of advanced technologies for enhancing assay performance.</p

Rapid, Simple and Inexpensive Fabrication of Paper-based Analytical Devices by Parafilm® Hot Pressing

Paper-based analytical devices have been substantially developed in recent decades. Many fabrication techniques for paper-based analytical devices have been demonstrated and reported. Herein we report a relatively rapid, simple, and inexpensive method for fabricating paper-based analytical devices using parafilm hot pressing. We studied and optimized the effect of the key fabrication parameters, namely pressure, temperature, and pressing time. We discerned the optimal conditions, including pressure of 3.8 MPa (3 tons), temperature of 80oC, and 3 minutes of pressing time, with the smallest hydrophobic barrier size (821 &amp;micro;m) being governed by laminate mask and parafilm dispersal from pressure and heat. Physical and biochemical properties were evaluated to substantiate the paper functionality for analytical devices. Wicking speed in the fabricated paper strips was slightly slower than that of non-processed paper, resulting from reducing paper pore size. A colorimetric immunological assay was performed to demonstrate the protein binding capacity of the paper-based device after exposure to pressure and heat from the fabrication. Moreover, mixing in two-dimensional paper-based device and flowing in a three-dimensional counterpart were thoroughly investigated, demonstrating that the paper device from this fabrication process is potentially applicable as analytical devices for biomolecule detection. Fast, easy, and inexpensive parafilm hot press fabrication presents an opportunity for researchers to develop paper-based analytical devices in resource-limited environments.</jats:p

Cell alignment and accumulation using acoustic nozzle for bioprinting

AbstractBioprinting could spatially align various cells in high accuracy to simulate complex and highly organized native tissues. However, the uniform suspension and low concentration of cells in the bioink and subsequently printed construct usually results in weak cell-cell interaction and slow proliferation. Acoustic manipulation of biological cells during the extrusion-based bioprinting by a specific structural vibration mode was proposed and evaluated. Both C2C12 cells and human umbilical vein endothelial cells (HUVECs) could be effectively and quickly accumulated at the center of the cylindrical tube and consequently the middle of the printed construct with acoustic excitation at the driving frequency of 871 kHz. The full width at half maximum (FWHM) of cell distributions fitted with a Gaussian curve showed a significant reduction by about 2.2 fold in the printed construct. The viability, morphology, and differentiation of these cells were monitored and compared. C2C12 cells that were undergone the acoustic excitation had nuclei oriented densely within ±30° and decreased circularity index by 1.91 fold or significant cell elongation in the printing direction. In addition, the formation of the capillary-like structure in the HUVECs construct was found. The number of nodes, junctions, meshes, and branches of HUVECs on day 14 was significantly greater with acoustic excitation for the enhanced neovascularization. Altogether, the proposed acoustic technology can satisfactorily accumulate/pattern biological cells in the printed construct at high biocompatibility. The enhanced cell interaction and differentiation could subsequently improve the performance and functionalities of the engineered tissue samples.</jats:p

Wicking in paper strips under consideration of liquid absorption capacity

Paper-based microfluidic devices have the potential of being a low-cost platform for diagnostic devices. Electrical circuit analogy (ECA) model has been used to model the wicking process in paper-based microfluidic devices. However, material characteristics such as absorption capacity cannot be included in the previous ECA models. This paper proposes a new model to describe the wicking process with liquid absorption in a paper strip. We observed that the fluid continues to flow in a paper strip, even after the fluid reservoir has been removed. This phenomenon is caused by the ability of the paper to store liquid in its matrix. The model presented in this paper is derived from the analogy to the current response of an electric circuit with a capacitance. All coefficients in the model are fitted with data of capillary rise experiments and compared with direct measurement of the absorption capacity. The theoretical data of the model agrees well with experimental data and the conventional Washburn model. Considering liquid absorption capacity as a capacitance helps to explain the relationship between material characteristics and the wicking mechanism.</p