Early detection of diseases can have a significant impact on patient outcomes. It can enhance the precision and efficiency of disease diagnosis if practical examples of the successful usage of diagnostic tools in developing nations are provided. Additionally, providing more information on the types of diseases that can be detected early using these tools can help readers understand the importance of early detection. It is important to consider the limitations or challenges associated with implementing these diagnostic tools in remote areas with limited access to medical care. This will provide a more balanced perspective on the potential implications of this technology and help readers understand the complexities of improving healthcare in emerging nations.
3. Design and Fabrication of Paper
The potential use of paper as a biosensor material is highly attractive owing to its flexibility, wide accessibility, economical nature, and hydrophilic properties. Its surface can readily be changed or shaped and has potent adsorptive capabilities for biomolecules and nanoparticles (NPs). Cellulose, the most common biopolymer, is a linear chain comprising several glucose units and the main ingredient of most paper kinds. Cellulose is a biodegradable fiber that is strong, non-absorbent, and water-insoluble 
The advantages of the paper include its non-toxic properties, easy disposal, and biodegradability. One of the primary advantages of cellulose is its porosity, which facilitates the capillary transport of solutions. Consequently, cellulose has the potential to function as a self-sufficient microfluidic pumping mechanism, avoiding the requirement for additional pumps. Paper’s inherent features (such as biocompatibility, porosity, flatness, and capillary forces) allow for novel developments in various applications. The affordability, ease of manufacture and operation, portability, and disposability of paper-based analytical equipment are all significant factors in their widespread use. The material’s adaptability renders it highly suitable for analysis decentralization, as demonstrated by its application in lateral and vertical flow devices 
, single or complex components, and its ease of integration with other flat materials such as tape. Additionally, this material can be manipulated through cutting, pressing, folding, or printing.
3.1. Paper Types Used in Electroanalytical Devices
Paper-based analytical instruments typically use cellulose fiber content-based materials or membranes made from nitrocellulose, two of the many paper types now available. Whatman® filter papers are widely used in the fields of chromatography and filtering because they are made from cellulose fiber (cellulosic paper). The most popular grade, no. 1, has moderate retention and flow rates. Most paper-based analytical devices are fabricated utilizing materials based on cellulose fibers or nitrocellulose membranes (Table 1).
Table 1. Summary of different types of paper substrates and their characteristics.
|Type of Paper Substrate
||RH Range (%)
|Filter paper (Whatman 4)
||Carbon black (CB) and
Reduced graphene oxide (rGO)
|Coating and drying
|Cellulose filter paper
||Soaking and drying
|A4 printing paper
||A4 printing paper
|A4 porous paper
|Graphite and silver
|Screen printing and
||1.59 (54 RH%)
63.7 (95 RH%)
|Metalized paper (aluminum-coated paper)
||Carbon nanotube and
||0.375 pF/RH% (30–70%)
8.24 pF/RH% (70–95%)
||Graphene printing ink
Paper-based analytical strips are cheaper to produce, more accessible to dispose of (they may be burnt), and need fewer operations (particularly, reagent-free devices). Due to the porous nature of filter paper, chemicals could be preloaded into the strips 
. Paper substrates often utilized for PAD fabrication include paper filters, blotting paper, and chromatography sheets of paper. Paper is a flexible, inexpensive, and widely available material. The development of PADs has extensively used Whatman type 1 chromatographic paper and the filter paper from Whatman no. 1. The materials that make up each paper generally consist almost entirely of cellulose (more than 98%). The advantages of using Whatman grade 1 chromatography are less expensive paper cost, high flow rate, consistent thickness, and good hygroscopic and wicking capacities. Both Whatman grade 4 papers for chromatography (pore size of 20–25 µM) and nitrocellulose (NC) paper have been utilized as substrates for protein immobilization due to their robust protein-binding capabilities, which are attributed to charge–charge connections and weak secondary factors, respectively. The paper-based sensors were fabricated utilizing diverse paper types, including chromatography paper from grade 3, paper towels, Whatman P81, and office paper. Although many different paper substrates are already in use, researchers are always on the lookout for more sustainable options.
Microfluidic channels, lateral flow 
, dipsticks, and origami designs are just a few of the many configurations for the instrument. The integration of origami-based paper sensors presents potential advantages in detecting volatile components, as it enables the absorption of vapor within the paper pores, followed by direct detection. Origami’s resistance to leaching and the coffee impact after reacting with analytes keeps the sensing assay’s rapid reaction times intact. Despite using various paper substrates in the construction of PADs, researchers continue to seek out substrates with distinctive attributes or locally produced through environmentally sustainable methods. Several methods are available for fabricating fluidic channels or assay zones on PADs, including inkjet printing 
, screen printing 
, wax printing 
, photolithography, plasma treatment, flexographic printing, wax dip, and laser cutting 
3.2. Fabrication of Paper-Based Electrodes
Several common methods exist for integrating sensing materials into paper substrates for paper-based biosensors. The choice of a suitable methodology depends on both the specific application scenario and the attributes of the sensing material. Some standard methods for integrating sensing materials into paper substrates include drop casting, inkjet 
, screen printing, spray coating, and electrodeposition methods 
3.3. Electrochemical Biosensors
Electrochemical biosensors are equipped with an identification component and electronic transducer, which facilitate the detection of highly sensitive analytes in human body fluids. Moreover, the high suitability of these devices for POC diagnostics is attributed to their portability, wearability, and implantability. These sensors include potentiometric, voltammetric, amperometric, photoelectrochemical, organic electrochemical transistors, and electro chemiluminescent sensors 
The electrochemical biosensor is a representative sensing apparatus that transduces biochemical occurrences into electrical signals. The current sensor design employs an electrode as a primary component, which functions as a reliable material for immobilizing biomolecules and promotes the transfer of electrons. Electrochemical biosensors present several benefits compared to traditional laboratory analytical methods, such as high-pressure liquid and gas chromatography combined with mass spectrometry. The primary attributes of these instruments encompass their capacity for mobility, ease of use, cost-effectiveness, quick analysis duration, elevated severity, and specificity within complex matrices 
. Numerous nanoparticles with broad surface areas improve loading capacity and reactant mass transfer for high analytical sensitivity 
. Recent years have seen an increase in the manufacturing of these biosensors, connected to the widespread utilization of nanomaterials such as graphene, its compounds, and nanocomposites (e.g., graphene oxides (GO) and CNT) 
Graphene, a hexagonal lattice structure, has garnered significant attention recently. Graphene has found utility in diverse biosensor uses, particularly in the field of electrochemical sensors 
. Graphene has identical fundamental physicochemical properties to graphite and carbon nanotubes, alongside its extensive surface area and numerous functional sites. Graphene’s exceptional electronic and mechanical characteristics make it suitable for label-free biosensors 
. Nanomaterials used as electrodes or facilitating matrices should have assisted electrocatalytic properties, excellent electron movement capacity, and remarkable biocompatibility with capturing biomolecules for signal amplification. Electrochemical techniques can be employed to incorporate nanomaterials into paper and microfluidic biosensors, thereby enabling the identification of biomolecules at the POC.
3.4. Improvement of ePAD Detection Performance by Nanomaterials
The employing of nanomaterials as electrode materials has the potential to expedite electron transfer and enable the effective loading of biomolecular explores and electrochemically reactive molecules 
. Therefore, nanomaterials can be used to fabricate electrodes for ePADs, and they can also be used to modify as-fabricated electrodes with nanomaterials to enhance their detection sensitivity. For improving the detection efficacy of the screen-printed carbon electrodes (SPCEs) in the ePADs, various nanoparticles were used, including noble metal (gold, platinum, and silver) nanoparticles, metallic oxides, and silica nanoparticles. Nanoparticles facilitate the rapid transfer of electrons among the transducer and analyte while also serving as covalent anchoring points for a variety of biorecognition probes. A recent study demonstrated that the carbon electrode in ePADs can effectively alter AuNPs, enabling electrochemically thiolated DNA probes to be constructed. The utilization of AuNPs in the fabrication of a carbon electrode on a paper substrate leads to an increase in both anodic and cathodic currents, as evidenced by enhanced charge, conductivity, and electrically charged area of the surface. This is attributed to the conductive properties of the AuNPs, which function as a conductive layer. It is observed that the ePADs are highly sensitive to the detection of microRNA 155 with a LOD of 33.8 nM 
. Furthermore, the paper-based carbon electrode enhanced with AuNPs displays remarkable stability after being stored for 60 days in the dark. Paper-based EC strips were produced by Cinti et al. 
, who utilized a drop-casting method to deposit AuNPs onto a carbon electrode made from paper, with the aim of detecting both single- and double-stranded DNA.
The utilization of AuNPs in immobilizing MB-tagged triplex-forming oligonucleotides (TFO) at the electrode surface and improving charge transfer kinetics is attributed to their distinctive characteristics, including favorable electrical properties, robust adsorption ability, facile attachment by covalent bonds to thiolated compounds, and elevated surface-to-volume ratio. Carbon nanotubes (CNTs), both single-walled and multi-walled, have been widely utilized as transduction components in electrochemical (EC) biosensors owing to their remarkable conductivity to electricity, expansive surface area, and varied surface-chemical properties. In addition, the utilization of paper coated with carbon nanotubes facilitates the production of nanostructures that possess porosity and a substantial surface area. Electrodes made from CNTs on paper have attracted considerable attention for use in ePADs. Valentine et al. 
deposited MWCNT-Nafion onto pre-cut sensing areas of chromatography paper using an ethanol suspension of the nanocomposites. The impact of paper pore size on the final MWCNT network and subsequent electrochemical sensing of glucose was investigated in this study. Tran et al. 
fabricated patterned single-walled carbon nanotube electrodes by vacuum filtering a layer of SWCNT onto a nitrocellulose (NC) membrane, which took approximately 15 min. The electrodes displayed notable conductive properties, using a mean resistance measuring below 100/sq, and exhibited remarkable mechanical durability when subjected to repeated bending assessments. AuNPs have the potential to be utilized for the development of non-enzymatic glucose ePADs through direct deposition onto single-walled carbon nanotube electrodes.
3.5. Designing and Fabricating 3D Paper Devices
The present methodology enables the construction of three-dimensional paper-based apparatuses through the utilization of folding, cutting, and lamination techniques applied to paper sheets, resulting in intricate geometric configurations. The paper devices are filled with active materials such as enzymes, dyes, and nanomaterials to create functional, low-cost, and sustainable devices. These devices are easy to produce, cost-effective, and eco-friendly. They have many applications, including medical diagnostics and sensing 
. Recently, Guan et al. 
employed glue adhesion to build a paper-based, three-dimensional analytical device with separate, specialized layers for separating plasma and detecting fibrinogen in blood samples and detecting linear values from 127.0 mg/dL to 508.0 mg/dL. In addition, the analytical device based on 3D origami does not require electricity or specialized instruments. It has the ability to detect HIV type 1p24, an infectious disease, at low levels as 0.03 ng/mL 
. The paper-based analytical device utilizing 3D origami has the capability to detect human immunoglobulin G (HIgG) at levels as low as 0.01 ng/mL 
Teengam et al. 
have devised a biosensor with high sensitivity for detecting M. tuberculosis
, demonstrating its potential for rapid and low-cost tuberculosis diagnosis. The biosensor utilizes the covalent immobilization of the pyrrolidinyl peptide nucleic acid (PPNA) on the 3D paper-based device, allowing for detecting M. tuberculosis
nucleotides. The findings revealed that the biosensor demonstrated a range of linearity of 2 to 200 nM and LOD value of 1.24 nM, indicating the potential for precise and sensitive detection of nucleotides associated with M. tuberculosis
3.6. Wearable Sensors
Paper-based electrochemical sensors that are wearable are a specific type of sensor that employs electrochemical techniques for sensing and paper as the substrate material. These sensors have gained popularity recently owing to their affordability, user-friendliness, and mobility 
. These sensors utilize paper that has been infused with conductive materials such as graphene, carbon nanotubes, or silver nanoparticles for the purpose of enabling electrochemical sensing. Additionally, a layer of enzyme or antibody is applied to the sensors to enable the detection of specific analytes 
. The flexibility and conformability of wearable paper-based electrochemical sensors render them advantageous for monitoring biological signals and health indicators on the skin. They can be integrated into wearable devices, such as smartwatches or fitness trackers, to continuously monitor biomarkers such as glucose, lactate, or cortisol. Additionally, using paper as a substrate makes them environmentally friendly and biodegradable. Wearable paper-based electrochemical sensors have a broad spectrum of potential applications in diverse fields such as temperature 
, pressure 
, light 
, humidity and respiration 
, and healthcare (monitoring of biomarkers, diabetes, or cardiovascular disease).