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To achieve rapid and simple extraction of nucleic acid, several papers (e.g., Fusion 5, glass fiber) have been used. However, the extraction efficiencies of these papers are still poor due to the nonspecific absorbance of cell debris and interferents (e.g., hemoglobin) on paper, which affects the extraction purity and sensitivity of downstream analysis. To enhance the extraction efficiency, several modification approaches have been utilized to change the surface charges of paper for nucleic acid extraction. For example, chitosan was used to modify Fusion 5 for paper-based DNA extraction (Fig. 5a) [12]. The device is composed of Fusion 5, PDMS membrane and PMMA. Fusion 5 was inserted into PDMS membrane and then sandwiched by two PMMA layers. In the modification process, Fusion 5 was modified sequentially by oxygen plasma activation, chitosan treatment, deionized water washing and vacuum drying. The capture principle of DNA in the modification process is based on the physical entanglement of long-chain DNA molecules and the fiber matrix and electrostatic adsorption between DNA and the chitosan-modified filter fibers. With this modification, the capture efficiencies of K562 human genomic DNA (up to 98%) and bacteriophage λ-DNA (up to 95%) are significantly improved. Fusion 5 can also preconcentrate λ-DNA from a diluted sample by more than 30-fold. But on the other hand, the extraction efficiency of nucleic acids of this device is affected by pH. The device requires syringe pump and valve to achieve automated extraction of DNA from a small volume of sample, indicating that it still needs improve its portability for bedside diagnosis. In addition, Whatman No. 1 filter paper was modified with spermine, polyvinylpyrrolidone (PVP) 40 and cationic polymers (e.g., polyethylenimine (PEI), dopamine, 3-aminopropyl trimethoxysilane (APTMS) and chitosan) to bind DNA/RNA from animal, plant and microbe samples (Fig. 5b) [109]. In the binding process, the surface of the modified filter paper has a positive charge and can bind negatively charged DNA by electrostatic adsorption. The binding ability of nucleic acids on the chitosan- and PEI-modified filter paper is better than that of other compounds. The cellulose dipstick is composed of a wax impregnated handle and nucleic acid binding region. The cellulose-based dipstick can extract nucleic acid within 30 second without any pipette or electrical equipment and can combine with simple portable amplification device for POCT applications. In short, these modification approaches for filter paper can improve the extraction efficiency of DNA and can be integrated into paper-based devices for the rapid testing of nucleic acids in POCT.
To address this issue, various fast, portable and inexpensive technologies for sample preconcentration have been developed. For instance, Whatman No. 1 filter paper modified with zinc oxide nanorods using a hydrothermal method was developed to fabricate paper-based ELISA device for preconcentration of myoglobin (Fig. 6) [13]. The ZnO nanostructured-paper composite can form the oriented micro- or nanostructures on paper to provide the high surface area of binding sites, which contribute to the immobilization of biomolecules. And the modified paper treated with 3-APTES through salinization can increase the antibody binding sites (amides) on the paper surface to capture the target protein (myoglobin) for protein preconcentration. This device can detect myoglobin based on the antigen-antibody interaction. And the ELISA testing result of this device indicates that the preconcentration efficiency of biomarkers from a diluted solution (myoglobin
To address this, different modification approaches have been developed to change the features of papers, including the surface functional groups, wet strength and specific surface area. For example, Whatman chromatography paper was modified with carboxymethyl cellulose (CMC) to form a linker for immobilizing biomolecules in a hydrophilic barrier made of wax as reaction region to promote the functionality and stability of paper-based ELISA devices (Fig. 7a) [122]. The device was used to detect tuberculin based on the antigen-antibody interaction, and its hydrophilicity increased by CMC can reduce non-specific binding of random proteins to improve its detection sensitivity. In the modification process, 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxy-succinimide (EDC/NHS) was crosslinked with the CMC-modified cellulose surface to produce NHS ester groups, which can enhance the specific binding between the paper substrate and the ligands or proteins by covalent conjugation. This modification technology can achieve a detection limit of 0.03 ng.mL-1 for tuberculin protein. The CMC-modified paper shows potential as an equipment-free diagnostic platform. However, the higher density of antibody attachment on paper substrate can increase the background noise and wax printer is expensive. Additionally, Whatman chromatography paper was modified with polyamidoamine starburst dendrimer (PAMAM dendrimer) to fabricate a paper-based device for the detection of telomerase activity (Fig. 7b) [123]. The modification approach functionalized the paper surface with amino groups, which can improve the efficiency of biomolecules immobilization. The paper-based device was fabricated by hand drawing with using a template and was used to detect telomerase activity based on the hybridization of Cy5 modified single strand DNA probes with telomerase extension products. The PAMAM-based device is a simple and amplification-free fluorescence assay, making it suitable for disease diagnosis at bedside. However, this modification is expensive. Additionally, a novel peptide of diphenylalanine (FF) was used to modify Whatman chromatography paper to prepare nanocomposite electrodes for detection of alpha-fetoprotein (AFP) (Fig. 7c) [124]. The device is composed of a lower sheet of plastic and an upper layer of cellulose paper modified with silver-graphene printed electrodes and uses antibody captured target protein to generate electrical response relevant to the concentrations of target protein. In the modification process, FF not only enhances the stability of immobilized antibodies via amine-aldehyde reactions, but also increases the wet strength of paper by forming paper-plastic integrated chips. The paper-based device can screen AFP with a range from 1 ng.mL-1 to 104 ng.mL-1 and achieve a detection limit of 10 ng.mL-1. It can be integrated into a miniaturized portable device as a paper-based biosensor for POCT. However, it still requires small amounts of reagents (e.g., hexafluoroisopropanol) compared to other methods.
To handle this problem, several modification approaches by changing the surface groups on papers have been developed and utilized to improve the performance of the paper-based devices. For example, cellulose paper was modified with 3-triethoxysilylpropylamine (APTES) and the prepared paper-based test strip was used to detect oxitetracycline (OXY) through observing color change (Fig. 10a) [132]. The modification method can aminate the paper surface using APTES based on the self-assembly approach, because the amine group can bind metal ions (Fe(III) and Cu(II)) to form color. The detection concentration of OXY using this device is as low as 30 ng.mL-1. This device is inexpensive, equipment-free and environmental-friendly, making it suitable for resource-limited setting. However, this modification process needs complex approach and is influenced by the reaction time of APTES. Furthermore, Whatman chromatography paper was modified with multiwalled carbon nanotubes (MWCNTs)/thionine (THI)/gold nanoparticles (AuNPs) nanocomposites, which function as the screen-printed working electrodes on paper for the detection of 17β-estradiol (Fig. 10b) [133]. The prepared paper-based device was used to detect 17β-estradiol based on electronic signal response. The modification process increases the specific surface area, which can improve the immobilization capacity of 17β-estradiol antibodies, allowing a higher density load of electroactive materials on paper surface to enhance the electrochemical current. The fabricated paper device has a wide linear detection range of 10 pg.mL-1 to 100 ng.mL-1 and a detection limit of 10 pg.mL-1. To achieve the really POCT, it still needs to develop a wireless portable electrochemical detector to combine with this device. However, the synthesis and functionalization of MWCNTs/THI/AuNPs is time consuming and complex.
To handle this problem, various modification approaches have been utilized to change the surface chemical groups and hydrophilic/hydrophobic features of paper for ion/chemical molecule detection. For instance, APTMS, 3-triethoxysilylpropyl succinic anhydride (TESPSA) and mercaptopropyl trimethoxysilane (MPTMS) were modified on Whatman chromatography paper by depositing vaporization for achieving multiplex detection of heavy metals (Fig. 11) [134]. The modification process immobilized amine groups, carboxyl groups and thiol groups on the surface of chromatography paper by condensation reactions. Three chromogenic reagents that reacted strongly with Ni(II), Cr(VI) and Hg(II) were covalently coupled to these functional groups, leading to color change of paper based on metal complexation reaction. The detection limits of the prepared devices are 0.24 ppm for Ni(II), 0.18 ppm for Cr(VI) and 0.19 ppm for Hg(II), respectively. It provides a simple and reliable analytical tool for POCT in low-resource settings. However, the uniformity of the modification approach need be further improved. In addition, filter paper modified with chitosan was also used to fabricate a colorimetric test strip for detection of mercury (Hg(II)) (Fig. 12) [29]. The paper test strip was fabricated by incorporating silver-doped CdS dots capped with mercaptoacetic into chitosan-coated filter paper, which can make a visualized color change from yellow to deep brown when Hg(II) ions were captured by the mercaptoacetic acid on CdSAg. The modification process enhanced the stability of CdSAg QDs immobilization and accelerated the process of colored adduct formation. The detection limit of the paper test strip for Hg(II) was 124 μM. It is a rapid and portable diagnostic, making it in real-time detection. However, this modification process is influenced by the chitosan concentration. Moreover, BSA was utilized to tune the wettability of Whatman chromatography paper and the BSA-modified chromatography paper was used as a substrate of patterned paper assay by simple writing and stamping patterns on its surface (Fig. 13) [135]. This modification technology achieved the position control and spatial confinement of assay regions via the low solubility of the colored product. And the fabricated paper assay was successfully used to perform multi-target detections of Cu2+ and Ni2+ based on the precipitation reaction. It provides a portable diagnosis tool for POCT. However, this modification process is affected by the reaction temperature. 2ff7e9595c
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