Photolithography is a complex technique that works by transferring structural geometry from a photomask and was the first method used for patterning paper-based microfluidic systems (Martinez et al., 2007). This process begins by coating the substrate with photoresist material. Next, a pattern is designed on the substrate (Figure 4E). The substrate is then cured, and the uncured photoresist is removed using a solvent, leaving behind a cured photoresist barrier (He et al., 2015c).

This technique can achieve the resolution with features as tiny as 100 μm or smaller (Gallatin and Liddle, 2024). This makes it excellent for designing complex devices. However, the process is time-consuming, involving several steps that can take hours to complete. Moreover, the requirement for extra instruments like a UV lamp, photomasks, spin coater, mask aligners, cleanroom facilities, hot plate, etc. can be problematic in terms of cost-effectiveness. While modern tools can speed up production for large-scale manufacturing, the cost is very high (up to millions) (Gallatin and Liddle, 2024). Furthermore, residue from the photoresist can cause colorimetric reactions and unwanted background (Dungchai et al., 2011). These expenses make photolithography less practical for small-scale projects or research with limited budgets. Despite these challenges, its precision and ability to handle large-scale production make it valuable for industries like semiconductor manufacturing. For μPADs, the high cost and complexity often outweigh its benefits in smaller projects.

Over the years, this technology has evolved from manual UV-lithography to faster alternatives such as FLASH (fast lithographic activation of sheets) (Martinez et al., 2008b), DMPC (dynamic mask photocuring) (He et al., 2015b), laser-based direct writing approaches (Songok et al., 2014), and utilizing the photocatalytic properties of TiO₂ to fabricate µPADs (He et al., 2015a). These advancements have simplified the technique further while saving time and money, making it more suitable for high-frequency prototyping and manufacturing.

Despite the advancements in photolithography for µPADs, some key considerations remain crucial for successful µPAD development using this method. The first step is selecting a photoresist compatible with both the paper substrate and the intended chemical or biological assays. Next, it is essential to ensure the photomask design accurately reflects the desired microfluidic patterns, as high-resolution masks are vital for precise patterning. Finally, choosing solvents that effectively remove uncured photoresists without compromising the paper or the cured patterns is critical for achieving reliable and high-quality µPADs.

Plasma treatment is another commonly used technique for creating microchannel patterns on paper substrates. Initially, paper is hydrophobicized using materials such as octadecyl trichlorosilane (OTS), hexamethyldisilane, or fluorosilanes (Li et al., 2021; Lim et al., 2019). Following this, plasma treatment is performed under vacuum conditions. During plasma treatment, specific regions of the paper become hydrophilic, making channels for fluid flow (Figure 4H). The most common plasma-treated µPADs are created using a sizing agent that makes the paper more water-resistant (like AKD-heptane) (Li et al., 2008).

This technique achieves moderate to high resolution in creating hydrophobic barriers and hydrophilic channels on paper substrates, enabling the production of well-defined microfluidic patterns. One of its key strengths lies in the rapid treatment times, typically requiring only a few minutes to complete the process, which enhances efficiency in device fabrication. However, the adoption of plasma treatment faces significant barriers due to its high initial equipment costs. The specialized plasma generators and vacuum chambers necessary for this process often come with a substantial price tag, ranging from several thousand to tens of thousands of dollars (Ozer et al., 2020). This high upfront investment poses a considerable challenge, particularly for smaller laboratories or startups with limited resources. Additionally, these systems are not compatible with organic solvents. Since many biological and chemical assays require reagents that involve organic solvents, this limitation might pose an issue (He et al., 2015c). Consequently, while plasma treatment offers excellent control over surface properties and produces thermally and chemically stable barriers, its commercial potential remains limited. The requirement for expensive, specialized equipment and the need for vacuum conditions during processing makes it less accessible for widespread adoption, especially in resource-constrained settings or mass production scenarios. As a result, plasma treatment is more likely to find its niche in specialized research environments or high-end applications where the precision and quality of surface modification justify the high equipment costs. Nevertheless, efforts have been underway to develop portable systems that can create microplasmas, offering cheaper and more flexible alternatives that can be used under ambient conditions (Kao and Hsu, 2014).

When patterning paper with plasma treatment, several critical factors must be considered to ensure optimal device performance. The intensity and duration of plasma exposure influence the depth and degree of surface change, while the presence of sizing agents or other chemicals in the paper can affect treatment efficiency. Manufacturing challenges may arise from excessively intricate channel designs, potentially leading to issues like clogging or uneven flow. It's essential to use mask materials resistant to plasma and compatible with clean patterning to maintain channel clarity. Moreover, the surface chemistry post-plasma treatment should be compatible with subsequent treatments, such as biomolecule immobilization for additional device functionalities.

The fusion of paper-based microfluidics with printing technologies represents an important step in µPADs fabrication (Li et al., 2012b; Xia et al., 2016; Tortorich et al., 2018). In the inkjet printing method, hydrophobic barriers are applied onto paper using specialized hydrophobic inks such as Alkyl Ketene Dimer (AKD), Methylsilsesquioxane (MSQ), and Hexadecenyl succinic anhydride (HSA) (Akyazi et al., 2018). Following printing, these inks are cured either through UV exposure or heat treatment, creating high-resolution hydrophobic barriers (Yamada et al., 2015) as shown in Figure 4G. Inkjet printing has numerous advantages beyond channel creation. It serves as a versatile tool for depositing a range of substances, including colorimetric indicators, metal precursors, enzymes, ionophores, pH buffers, masking reagents, and blocking reagents, into both sensing and pretreatment regions (Tenda et al., 2016). This method ensures better accuracy in reagent volumes than manual pipetting techniques. Moreover, it facilitates accurate positioning and spatial confinement of deposited reagents, which is crucial for optimizing device performance. The continuous evolution of inkjet systems, such as Piezoelectric inkjet printing (e.g., Epson) and Thermal inkjet printing (e.g., Canon, Hewlett Packard), has further enhanced the capabilities and efficiency of this fabrication approach (Maejima et al., 2013; Yamada et al., 2015).

Inkjet printing is one of the most effective methods for developing μPADs, offering high resolution in the range of 50–200 μm. This level of precision enables the creation of detailed channel designs and intricate patterns, making it ideal for a variety of analytical applications. In terms of speed, inkjet printing is particularly efficient for small-scale production. Budget inkjet printers can achieve output rates of up to 15–25 pages per minute, making them well-suited for rapid prototyping and small-batch manufacturing. The technique is also cost-effective, striking a balance between affordability and functionality. Entry-level inkjet printers suitable for μPAD fabrication are available for as little as $30 to $100, while more advanced models for specialized tasks can cost up to $250. Operational expenses are reasonable as well, with replacement ink cartridges typically priced between $10 to $35. This combination of precision, speed, and cost-efficiency makes inkjet printing a versatile and accessible option for μPAD production.

When fabricating µPADs via inkjet printing, some factors must be considered. One concern is the potential formation of crystals or solid deposits at the printer nozzle, which can restrict the range of reagent concentrations feasible for use. Additionally, volatile reagents may evaporate too quickly, necessitating the use of less volatile alternatives (e.g., substituting cyclohexane for tetrahydrofuran). Another issue is the slow curing process, which can lead to low pattern resolution due to the spreading of reagents or hydrophobic ink. A potential solution for µPADs that require UV curing after printing is to immediately freeze them post-printing.

Since 2009, wax-patterned μPADs have been a popular choice. In these devices, the paper substrate is first patterned with wax to create channels that guide fluids. This initial patterning is followed by a heating step (using an oven, hot plate, or other hot surface) that melts the wax and allows it to penetrate the paper fibers, creating hydrophobic barriers that define hydrophilic channels (Dungchai et al., 2011). After cooling, the solidified wax creates the final design as shown in Figures 4A,D.

A key advantage of this method is the elimination of organic solvents during fabrication. Furthermore, wax patterning offers versatility, with techniques ranging from simple wax pens to commercial wax printers, screen printing, wax dipping, and even 3D printing of wax onto the paper surface (He et al., 2015c). The resolution achievable with wax printing ranges from hundreds of micrometers to centimeters, making it suitable for many analytical applications. This technique is highly efficient, offering fast production speeds ideal for mass manufacturing. Wax printing requires relatively low-cost wax materials, making it a cheaper alternative for resource-limited settings. Its simplicity and scalability provide high commercial potential, particularly for producing disposable μPADs for environmental or healthcare monitoring. However, commercial wax printers have been discontinued, so researchers are encouraged to explore alternative options such as thermal transfer printing, laser cutting, screen printing, photolithography, and laminate capillary-driven microfluidics (Ruiz et al., 2022; Le et al., 2017; Chiang et al., 2019).

Many researchers use laser treatment in the making of µPADs (He et al., 2015c). The technique uses a CO₂ laser or such laser to create or remove material from the paper substrate. Using a laser’s power, speed, and focus, channel depth and width can be controlled. The laser creates these channels by vaporizing the material off the paper. As a result, the unetched areas stay hydrophilic, allowing liquids to flow through and giving the device its microfluidic properties (Ghosh et al., 2019; Sousa et al., 2024). In 2011, laser treatment was first adapted for fabricating μPADs by using a CO₂ laser cutter to treat the surface of parchment paper (Chitnis et al., 2011).

Laser treatment technique achieves high resolution, capable of producing features smaller than 100 μm, which is particularly advantageous for applications requiring intricate channel designs or fine control over fluid flow. The process is relatively fast, especially for small-scale production, allowing for rapid prototyping and iteration of device designs. While the initial investment in laser equipment can be moderate to high, the operational costs are generally low, and the method offers good repeatability. The commercial potential of laser treatment is particularly notable for specialized applications that demand high-resolution features or complex geometries. Its non-contact nature and ability to process various paper substrates make it adaptable to different μPAD designs.

Recent advances in fabricating microfluidic paper-based analytical devices using a laser printer (LP-µPAD) have harnessed the precision of xerography-based commercial laser printers to create high-resolution microfluidic devices on paper (Ghosh et al., 2019). This direct printing procedure automates the deposition of hydrophobic toner ink, eliminating human interference and fabrication irregularities (Ghosh et al., 2019). Moreover, utilizing silver nanowire (AgNW) ink patterned with a solvent-free transfer method is also a promising alternative (Huang et al., 2016).

In addition to these popular methods, various techniques have evolved over the years for the fabrication of µPADs. One commonly used technique is flexographic printing (Figure 4C), where hydrophobic barriers or inks are printed on paper using volatile organic compounds or polystyrene ink (Olkkonen et al., 2010). This is one of the fastest µPAD fabrication techniques but struggles with design flexibility. It also requires multiple printing optimization methods to achieve higher resolution, making it less suitable for lab prototyping but holding potential for commercial manufacturing.

Cutting and shaping to create 2D and 3D µPADs have also been adopted to develop instrument-free fabrications (Figure 4B). This technique involves using handheld blades, biopsy punches, knife cutting, laser cutting, etc. (Zhong et al., 2024). It is economical and advantageous because it does not require any chemical-laden instrumentation. Since no wax, organic solvents, or inks are used, there is a better chance of achieving higher resolution. However, the technique is limited to specific designs because boundaries cannot be defined as precisely as with wax or solvent lining.

Wet etching is another method, where hydrophobic paper is etched with a specific pattern (Cai et al., 2014). It starts with preparing the substrate, which includes cleaning and putting a protective mask or pattern (e.g., trimethyloctadecylsilane (TMOS)) on it. This mask defines the areas that will be unaffected by etching. The substrate is then immersed in an etchant solution (e.g., NaOH), which selectively reacts with the exposed portions based on the mask design. The etchant dissolves or changes the substance in these locations while leaving the masked sections unaffected, creating microfluidic channels. After the desired etching time, the mask is normally removed, and the substrate is cleaned to eliminate any remaining chemicals.

Stacking and origami techniques are also useful methods for developing µPADs (Kaewarsa et al., 2021; Das et al., 2019). These techniques enable multi-step chemical and biological assays. Folding, sliding, and rotating can be used to automate multiple steps in these systems (Carrell et al., 2019; Yakoh et al., 2022; Verma et al., 2018). Furthermore, chemical vapor-phase deposition, corona treatment, chemical modifications, stamping, plotting (Figure 4F), and lacquer spraying methods have also been employed to develop µPADs (Demirel and Babur, 2014; Jiang et al., 2016; Curto et al., 2013; Liu et al., 2017; Shakeri et al., 2019). Figure 4 shows the common fabrication methods for creating patterns on paper-based microfluidic devices.

Table 1 shows the summary of fabrication methods for µPADs. This table compares various techniques based on their suitability for mass production, ease of use, and resource requirements. Among these methods, wax printing, inkjet printing, and flexographic printing show the most promise for commercial applications due to their balance of resolution, speed, and cost-effectiveness. Laser treatment is emerging as a versatile method for high-resolution fabrication. Photolithography, while offering high resolution, faces challenges in large-scale adoption due to its complexity and cost. Screen printing remains relevant for certain applications due to its simplicity and moderate resolution. Plasma treatment, plotting, and wet etching have more limited commercial potential due to various constraints in scalability or process complexity. Therefore, for large-scale production, techniques such as inkjet and flexographic printing are recommended, while more accessible methods like wax printing and plotting are ideal for beginners with limited resources.

In addition to the aforementioned factors, the choice of recognition elements for μPADs is crucial, as their compatibility with the fabrication method directly affects the device’s sensitivity and specificity. For wax printing, thermally stable and polar recognition elements are ideal. Reagents requiring organic solvents like DMSO or chloroform should be avoided, as they can dissolve the wax barrier. Photolithography benefits from UV-resistant and stable materials. For screen printing, recognition elements that integrate well with conductive inks are ideal. Recognition elements that are heat-tolerant and stable under high-energy laser processing are essential for laser-treated μPADs. For inkjet printing, recognition elements compatible with low-viscosity ink formulations are essential. Additionally, the elements should be stable in liquid form and maintain their functional properties after deposition. For plotting, elements that can interact effectively with hydrophilic surfaces are ideal. For wet etching, recognition elements must be compatible with chemical solvents and porous substrates, remaining stable in the presence of acids or bases and retaining their functionality throughout the etching process. Most of these physical properties of elements are considered if they are pretreated on substrates before the fabrication process. On the other hand, chemical properties such as solvent compatibility, stability, and potential interactions with the hydrophobic barrier are crucial factors that need to be carefully studied before using a fabrication technique, regardless of pre- or post-treatment.

Colorimetry is a commonly used method to generate signals in µPADs because the analyte concentration can be directly or indirectly correlated with the color intensity. Additionally, the color can be quantified using various portable platforms. There are numerous established colorimetric chemistries, including metal-ligand complexation, enzyme-inhibition assays, acid-base indicators, and redox indicators (Yamada et al., 2017a). Often, these signals are distinguishable enough to allow for semi-quantitative and qualitative observations, as seen in pregnancy tests, pH strips, and home water test kits (Morbioli et al., 2017).

Advancements in colorimetric detection have led to innovations ranging from simple spot tests to distance-based detection, where the distance of the colored signal is proportional to the analyte concentration, making it a valuable tool for semi-quantitative analysis (Cate et al., 2015; Cate et al., 2013). Furthermore, other methods such as time-based quantitative color readouts, counting-based readouts, barcode-like readouts, text-displaying readouts (Figure 5A), and direct result readouts have been developed over the years (Karita and Kaneta, 2014; Guan et al., 2014; Li and Macdonald, 2016; Lewis et al., 2012; Lewis et al., 2013; Zhang et al., 2015; Li et al., 2012a). These innovative techniques enable calibration-free assays, enhancing the utility and accuracy of colorimetric detection in paper-based microfluidics.

While colorimetric systems are simple and effective, they do have drawbacks. A significant issue in quantitative colorimetric systems is the non-homogeneity of color (Morbioli et al., 2017; Yan-Qi and Liang, 2020). In µPADs, colored indicators used for detection can migrate unevenly, leading to patchy color development. This happens because highly soluble signaling molecules spread outwards during the sample flow, accumulating at the edges of the designated detection zone. This issue can be tackled by employing attachments, either covalent or non-covalent, directly onto the paper itself (Tan et al., 2022). Another major hurdle is the inconsistency of lighting. Initially, colorimetric systems were imaged using scanners, which provided higher resolution and consistent lighting. However, for Point-of-Need (PoN) or Point-of-Care (PoC) devices, it is ideal to have systems that are instrument-free or can be quantified using easily accessible tools like smartphones. Constructing a lightbox for imaging is one solution to this issue, allowing the use of a smartphone camera. Introducing a background region of interest (ROI) is another way to mitigate this problem, as it helps normalize intensities under varying lighting conditions (Tan et al., 2022; Aryal et al., 2024d; Richardson et al., 2021). Recently, third-party apps for color quantification have become available in app stores that have an onsite calibration feature built into them. Another challenge arises if the analyte itself is colored. In such cases, using a color control can help mitigate this issue. While distance-based semi-quantitative signal readouts allow for calibration-free assays, they are susceptible to observer-dependent readout errors. Counting-based semi-quantitative signal readouts are less prone to observer-dependent errors and can achieve calibration-free assays, but they have relatively limited resolution and are often time-dependent. Similarly, timing-based signal readout systems face the same limitations. Text-based detection platforms are often restricted by the target analytes that can be used for detection, as they may not be applicable for every assay (Yamada et al., 2017a).

Electrochemical paper-based analytical devices (ePADs) are among the most used µPADs after colorimetric µPADs. This technology offers several advantages. Firstly, ePADs leverage external detectors like potentiostats, enabling them to achieve significantly lower detection limits, reaching the picomolar range (Aryal et al., 2024b; Adkins et al., 2015; Oh and Chow, 2015; Mettakoonpitak et al., 2016). This surpasses the capabilities of techniques like fluorescence and colorimetry (Pohanka and Skládal, 2008; Dungchai et al., 2009; Liu et al., 2014a). Additionally, ePAD results are unaffected by color variations within the sample itself, unlike colorimetric assays which can be prone to misinterpretations due to background color (Dortez et al., 2024). ePADs exhibit minimal impact from variations in sample volume as long as the designated electrode area is adequately covered (Yamada et al., 2017a). This provides greater flexibility compared to colorimetric methods where precise volume control is often critical. Moreover, electrochemical measurements can be performed relatively quickly, taking only seconds to minutes. This speed is especially advantageous when handling many samples (Noviana et al., 2020b). The conventional configuration for an electrochemical system consists of three electrodes: a working electrode, a counter electrode, and a reference electrode (Adkins et al., 2015; Dungchai et al., 2009). Since the development of the first ePAD in 2009 by screen printing carbon ink on paper (Dungchai et al., 2009), many other methods have been developed over the years. These methods include stencil printing, inkjet printing, microwire placement, laser scribing, using carbon tape, pencil drawing, spray and spin coating, laser scribing, and sputtering (Noviana et al., 2020b; Ataide et al., 2020; de Araujo et al., 2017). Figure 5B shows an example of innovative electrochemical sensing on paper using 3D capillary-driven sequential microfluidics.

While electrochemical µPADs outperform colorimetric paper devices in terms of analytical performance owing to the usage of external detectors (e.g., potentiostats), due to their high cost (usually > $1000) and operating complexity, laboratory-type electrochemistry stations are inaccessible to end consumers. The most important problem for the real-world deployment of ePADs is to build user-friendly signal detection interfaces. User-friendly signal detection interfaces allow for real-time data analysis and easy-to-understand data displays, helping users make quick decisions. To bridge the gap, many handheld potentiostats have been developed that allow for onsite/remote sensing. Some notable examples of portable potentiostats integrated with μPADs include inkjet-printed paper-based potentiostats (Bezuidenhout et al., 2018), USB-controlled potentiostats (Chhorn and Teeramongkonrasmee, 2018), battery-less NFC potentiostats (Chaiyo et al., 2024), and Wi-Fi cloud-based portable potentiostats (Bianchi et al., 2020). Among these portable systems, the wireless NFC potentiostats hold great promise. These potentiostats utilize near-field communication (NFC) for wireless power and data exchange with a user-friendly smartphone app (Krorakai et al., 2021). These portable and cost-effective systems offer analytical capabilities on par with traditional benchtop potentiostats (Beck et al., 2023). These potentiostats have been applied in μPADs-based electrochemical sensing of analytes like cholesterol and glucose and in performing various electrochemical lateral flow assays (Chaiyo et al., 2024; Gonzalez-Macia et al., 2024; Promsuwan et al., 2023; Lazaro et al., 2023). However, compared to benchtop potentiostats, portable potentiostats usually have higher noise levels, a smaller voltage range, and lower accuracy. Because of these drawbacks, portable potentiostats may not be as ideal for uses needing a broad voltage range or high precision and require further research and development (Aryal et al., 2024b).

Researchers using ePADs may face challenges such as restricted mass transfer, electrode fouling, a limited dynamic range, and inconsistent performance with disposable electrodes. To address these issues, smaller channels and increased flow rates can be employed to enhance mass transfer and allow analyte molecules more time to interact with electrode surfaces. To mitigate electrode fouling and improve electron transfer in electrochemical sensors, various strategies can be explored. These include employing nanoengineered surfaces like nanoporous metals and nanocarbons, applying antifouling layers such as PEG, zwitterionic polymers, and biopolymers, utilizing nanoporous membranes, and integrating hydrogels (Russo et al., 2021). In certain instances, the limited dynamic range issue can be resolved by expanding the electrode surface area (Arroyo-Currás et al., 2017). The use of ePADs coupled with wireless technology has been increasing and holds promising potential for the future (Solhi et al., 2020; Ruecha et al., 2017; Deroco et al., 2023; Fan et al., 2017). This method enables the wireless acquisition of data, which can be utilized for continuous monitoring in various fields such as sports, healthcare, agriculture, and defense. It allows users to receive alerts in case of abnormal situations and to be warned before these situations worsen. However, one challenge that wireless ePADs face in terms of sensing is the lifespan of the signals (Solhi et al., 2020). Researchers are encouraged to work on integrating wireless and remote sensing technologies to make this system more robust and reliable.

Chemiluminescence (CL)-based paper microfluidics (μ-PCLDs) have also gained popularity, particularly in CL-immunoassays. CL works on the principle of generating photons through a chemical reaction, and these photons are typically quantified in the dark to avoid non-specific and background signals (Calabretta et al., 2021). Figure 5D shows an example of a chemiluminescence based detection of cotinine in serum using a μPAD. With advancements in noise control modifications, this method has become more sensitive and specific, allowing for more accurate routine biomedical and health monitoring applications (Ge et al., 2014). μ-PCLDs can be tailored to achieve a broad dynamic range, depending on the detection target. They can detect analytes like uric acid and glucose at millimolar concentrations while also being capable of detecting DNA at molar levels (Wang et al., 2013c; Yu et al., 2011b; Yu et al., 2011a). CL on paper is more efficient than typical CL tests because it consumes fewer samples and reagents, shortens analysis time, and uses automated, compact, and integrated detection techniques (Aryal et al., 2024b). Additionally, the development of antibody-based probes has enabled these systems to detect antigens at trace levels, even in complex matrices (Calabretta et al., 2021). Over time, CL-based microfluidics have been employed for detecting tumor markers, DNA, antibodies, and other biomolecules (Yamada et al., 2017a).

However, there remains a significant gap in translating μ-PCLDs from lab to practical use. This gap is primarily due to the need for expensive instruments, such as luminescence readers. While μ-PCLDs offer incremental gains in sensitivity compared to colorimetric µPADs, these gains are often overshadowed by the exponential increase in operational costs. Despite this, the development of CMOS sensors, which are less complex and less expensive than conventional instruments, offers a promising solution (Punjiya et al., 2014; Hu et al., 2020; Khan et al., 2018).

Fluorescence based methods are also used with µPADs. The unique properties of fluorescent materials enable the creation of these assays. Tiny fluorescent particles, such as rhodamine, pyrene, or anthracenes, are deposited onto the paper (Nawaz et al., 2021). These particles then interact with the target molecule of interest. When ultraviolet light of a specific wavelength shines on the paper, any bound analyte triggers the fluorescent particles to emit light (Patel et al., 2021; Lian et al., 2020). The intensity of this emitted light, captured with a smartphone or scanner, can be analyzed using software like ImageJ to quantify the amount of target analyte present (Patel et al., 2021). Figure 5C shows an example of ratiometric sensing with fluorescence detection and digital readout.

µPADs using fluorescence measurements face more limitations compared to other detection methods. First, the paper itself can introduce noise by reflecting the excitation light, leading to a lower signal-to-noise ratio compared to techniques like electrochemiluminescence (Yamada et al., 2017a). Additionally, when using non-pure cellulose paper for convenience, the presence of optical brightening agents can further disrupt the assay by causing unwanted background fluorescence (Kwon et al., 2012). Some fluorescent molecules are unstable on paper across time and varying assay conditions, especially in fluctuating temperature and humidity environments. Use of stabilizing buffers or chelating agents can be used to enhance their stability.

Electrochemiluminescence (ECL) is a light emission process that occurs when chemical species generated through oxidation-reduction reactions undergo exothermic reactions, emitting light energy. Depending on the source of radicals, there are two main ways electrochemiluminescence (ECL) can occur. In one method, light is produced when a single molecule undergoes a reaction that creates energetic particles. In the other method, light is generated by the interaction between a special molecule and another molecule in the solution (Chinnadayyala et al., 2019).

ECL-µPADs have gained popularity in healthcare applications, particularly for PoC diagnosis and disease monitoring. However, due to the electrochemical nature of the process, most luminophores in ECL are less soluble in water, making it challenging to create binding sites without affecting their chemical properties. This presents a significant hurdle in designing µPADs with ECL readouts, often resulting in compromised sensitivity compared to electrochemical methods. To address these challenges, various signal amplification strategies have been developed over the years. One promising advancement is the incorporation of nanomaterials, which can act as electrocatalysts in ECL reactions. These nanomaterials promote electron transfer at the electrode interface and increase the loading capacity of ECL labels. Common labels include carbon dots, inorganic complexes, semiconductor nanocrystals, graphene quantum dots, and photoluminescent carbon nanocrystals (Chinnadayyala et al., 2019). While paper-based ECL biosensors have seen progress in research labs, their commercialization also remains a challenge. A crucial step towards practical application involves a comprehensive understanding of key design aspects for ECL paper devices. This includes factors like detection limits, specificity, assay speed, analysis format, compatibility with various sample types, packaging considerations, labeling requirements, shelf life needs, and target production costs (Chinnadayyala et al., 2019).

Other methods for signal detection in μPADs include photoelectrochemical detection (Figure 5F), surface-enhanced Raman scattering (SERS), digital microfluidics on paper, calorimetry, dual mode detection (Figure 5E), and light-reflectance (Davaji and Lee, 2014; Abbas et al., 2013; Liu et al., 2014b; Fang et al., 2014; Fobel et al., 2014). In photoelectrochemical (PEC) detection, the process is reversed compared to electrochemiluminescence. Instead of light triggering a chemical reaction, chemical light emission from an internal system, such as luminol-hydrogen peroxide, is converted into an electrical current (Zhao et al., 2016). This current is then detected by a semiconductor material, such as quantum dots. Researchers have used PEC as a labeling technique for various immunoassays, including those targeting tumor marker proteins, ATP (adenosine triphosphate), and DNA (Wang et al., 2013a; Wang et al., 2013b; Ge et al., 2013). Digital microfluidics (DMF) technology offers a powerful tool for handling tiny liquid droplets, ranging from nanoliters to microliters in volume (Choi et al., 2012). This technique uses an array of electrodes and electrical fields to manipulate these droplets. By controlling the electrical fields applied to the electrodes, functionalities such as merging separate droplets, splitting a single droplet into smaller ones, mixing different droplets, and directing the flow of droplets across the surface can be achieved (Choi et al., 2012). This technique is an exciting addition to sensing on μPADs. Table 2 compares analytical methods for detection on μPADs, showcasing their strengths and weaknesses. Figure 5 depicts common sensing methods for μPADs.

The integration of smartphones with microfluidic paper-based analytical devices (μPADs) has significantly advanced their capabilities, transforming them into portable, user-friendly diagnostic tools ideal for point-of-care applications and resource-limited settings. Lately, colorimetric paper-based platforms have seen a significant advancement. A few notable examples include the following: a novel smartphone-based solution with a color correction algorithm to achieve accurate and absolute color measurements (Zhang et al., 2023), the development of smartphone applications for color quantification (Shen et al., 2012; Wu et al., 2024), and the development of a custom app to analyze specific μPAD regions to provide accurate concentration data (Liu et al., 2024). For electrochemical μPADs, miniaturized potentiostats connect to smartphones via audio jacks or data ports, enabling real-time control, data processing, and signal analysis directly on the smartphone screen (Beck et al., 2023). Fluorescence-based μPADs have also seen notable advancements, A notable advancement is the smartphone-integrated colorimetric sensor array and fluorometric detection system, which uses image processing algorithms to quantify fluorescence intensity (Chellasamy et al., 2021). Together, these innovations make μPADs increasingly sophisticated and accessible for on-site, real-time diagnostics without the need for complex laboratory equipment.

While urbanization is increasing globally, a sizable proportion of the population in emerging countries still lives in rural areas (Desa, 2014). Even though poverty is a difficult concept to define, many people in developing countries rely on subsistence activities for a living, which significantly impacts their health (Yetisen et al., 2013; Sociales, 2009). These resource-limited environments frequently lack the technical skills and staff available in urban areas, making access to healthcare difficult. Affordable technologies are critical for empowering these local populations and improving their healthcare access. This is where μPAD technologies can be particularly beneficial. While these technologies are more urgently needed in developing nations, where even basic illness or disease diagnosis can be challenging, recent pandemics have also underscored the importance of PoC sensors in developed countries as well. This demonstrates the broad applications and usefulness of μPAD technologies across various global contexts. A prime example of the importance of these devices emerged during the COVID-19 pandemic. On 30 January 2020, the World Health Organization (WHO) declared the COVID-19 pandemic a global health crisis (Ravi et al., 2020; World Health Organization, 2020). Since the onset of the pandemic, a significant number of paper-based systems for COVID-19 diagnostics have been developed and published (Pinheiro et al., 2021; Kim and Lee, 2022; de Araujo et al., 2024). Long before and throughout the COVID-19 pandemic, researchers have actively explored using paper-based platforms to create biosensors for diagnosing various diseases and infections (Silva-Neto et al., 2023). These devices offer a promising and versatile approach to healthcare diagnostics. Here are some examples of μPADs for diagnostics and health monitoring:

Pollution is a major issue worldwide, affecting the health of people in developed and developing countries. Pollution can be of greater concern in developing countries, where large populations suffer from industrial waste, poor hygiene, unsafe drinking water, and unclean indoor air caused by burning organic matter due to the lack of established and enforced regulatory policies. The problem worsens in these areas because they lack affordable and fast ways to test for pollution/toxins. The progress of industry in developed countries has also created new types of pollution that can spread quickly and overwhelm the environment’s natural ability to clean itself. While traditional methods for monitoring environmental contaminants are reliable, they suffer from being time-consuming, expensive, and require specialized personnel. These limitations render them unsuitable for on-site testing. Therefore, researchers have increasingly focused on μPADs as a promising alternative for the detection of a wide range of contaminants, including heavy metals, pesticides, nutrients, microorganisms, and emerging threats like PFAS (per- and poly-fluoroalkyl substances) and particulate matter. We recently published a critical review on microfluidics for environmental monitoring, encompassing all the latest advances in the field (Aryal et al., 2024b). In this section, we will only focus on the general trends and gaps observed in μPADs for environmental monitoring.

The use of μPADs in environmental analysis spans various matrices, such as water, soil, air, agriculture products, etc. The most targeted contaminants are heavy metals, pesticides, nutrients, and per- and polyfluoroalkyl substances (PFAs) (Aryal et al., 2024b). Among the detection techniques employed, colorimetric methods are the most prevalent due to their simplicity and cost-effectiveness as well as their ability to be used onsite (Patel et al., 2021; Aryal et al., 2024a). Most applications have also focused on water samples. This is because water’s natural fluidic properties, such as low viscosity and surface tension, enable consistent flow and efficient mixing within the microchannels, ensuring precise sample processing (Saez et al., 2021; Atencia and Beebe, 2005). In contrast, for soil or air matrices, the samples need to be pretreated to get them into fluids, or complicated detection methods must be used to accommodate the differences in sample properties. In soil matrices, μPADs are primarily used for detecting nutrients, microorganisms, and other inorganic contaminants. The detection techniques used in soil analysis are more varied compared to those in water-based systems where colorimetric detection is very common. This diversity arises because soil samples require pretreatment and clarification before colorimetric analysis, as the natural color of the soil can interfere with the results. In addition to detecting contaminants, μPADs in soil are also used to monitor the behaviors of microorganisms, including their growth patterns, metabolic activities, and interactions with soil components. For air matrices, the primary focus is on detecting particulate metals and airborne pathogens. The most common techniques in air analysis are electrochemical and colorimetric methods. Figure 6C shows an innovative use of μPAD utilizing gold nanomaterials for the simultaneous quantification of Gram-negative bacteria and nitrite ions in water samples.

Detecting contaminants using μPADs in complex matrices like soil, seawater, and air remains challenging. Factors such as topography, temperature, and humidity can further complicate the measurement in these matrices. Therefore, when designing μPADs for complex environmental matrices, it is not possible to create a perfect miniaturized copy of a real soil or air environment within a microfluidic chip. Instead, these devices should focus on mimicking specific aspects of soil or air under controlled conditions. Moreover, when designing these tools, scientists need to clearly define which soil or air features they aim to replicate, and their findings should reflect the specific conditions they mimicked. μPAD-based PoN sensors are a promising development for environmental monitoring; however, many of these quick tests often rely on qualitative colorimetric methods to indicate results. Such methods can be subjective to the reader/user. Alternatively, techniques like EChem or fluorescence require external power and additional equipment. Therefore, there’s a critical need to develop quantitative/semi-quantitative colorimetric methods specifically for PoN applications. This is important because the majority of contamination occurs in resource-limited settings where sophisticated quantification resources may not be readily available.

Ensuring the safety of food is a top priority for both the food industry and those involved in agriculture. This means in the food industry, thorough checking for biological hazards, chemical contaminants, and allergic substances is essential to ensure food safety. Similarly in agriculture, regular monitoring of pesticide content, nutritional content, and potential health risks from cultivation to harvest is crucial for producing safe and healthy food. μPADs are increasingly being used to identify various contaminants in foods and beverages due to their portability and simplicity (Nilghaz et al., 2021). These devices provide a quick and easy solution for food safety and quality control in a variety of situations, including food processing plants and residences (Mitrogiannopoulou et al., 2023). They are used to identify foodborne pathogens like E. coli and Salmonella and pollutants like pesticides and heavy metals (Ji et al., 2020). They are also used to detect food spoilage, evaluate food quality indicators, and even analyze nutritional content (Hua et al., 2018; Soman et al., 2024; Wang et al., 2022). Figure 6B shows an innovative μPAD-based method of visual and real-time monitoring of Cd²⁺ in rice using [2+2] lanthanide clusters.

Detection of contaminants and microorganisms in food and beverages using μPADs involves several sensing methodologies depending upon the detection limit required and complexity of sample pretreatment. For bacterial detection, enzymatic activity or immunoassays are frequently employed (Nadar et al., 2021). These methods rely on colorimetric detection based on enzyme reactions with chromogenic substrates or electrochemical detection based on redox reactions involving the transfer of electrons between the bacterial metabolites or immunological markers and the electrode. To detect heavy metal contaminants like lead (Pb) and cadmium (Cd), electrochemical methods are commonly used (Busa et al., 2016). The most commonly used method for pesticide detection involves enzymatic inhibition by the pesticide, with color or an electrochemical signal used as the readout (Nadar et al., 2021; Hefner et al., 2024). Additionally, for detecting some other pesticide contaminants, methods like electrochemiluminescence (ECL) and chemiluminescence are employed (Busa et al., 2016). Even after years of advances, the promise of μPADs for food quality and safety analysis remains largely unfulfilled due to limitations in user-friendliness and sensitivity. The majority of food samples need to be pretreated before using them in μPADs. Therefore, more research on integrating on-chip sample pretreatment is needed.

In critical security concerns such as terrorist attacks, chemical spills, biological warfare incidents, radiological emergencies, hazardous material accidents, fire, and explosion accidents, swift detection of contaminants is crucial. In the aftermath of an incident, it's essential to quickly screen numerous people and surfaces for harmful substances before sending samples to a lab for confirmation. Similarly, at high-traffic checkpoints, rapid tests are necessary to detect harmful chemicals and prohibited drugs that might be smuggled. This is where μPADs can provide prompt results that save time and enable security officials to make timely decisions. Paper-based microfluidics have the potential to be utilized in international security systems for the detection of various prohibited chemicals, warfare agents, and explosives. They are employed in screening luggage, packages, and suspicious materials at checkpoints. Common targeted chemicals include peroxide explosives like triacetone triperoxide (TATP) and diacetone diperoxide (DADP), as well as nitroaromatic explosives such as trinitrotoluene (TNT), picric acid (PA), and dinitrotoluene (DNT) (Gökdere et al., 2019; Tian et al., 2017; Pramanik et al., 2019). μPADs are also used to detect illegal drugs in the field, thereby supporting law enforcement efforts (Noviana et al., 2020a).

In forensic analysis, presumptive tests serve as rapid initial screenings that help investigators detect potential substances. However, these tests have significant drawbacks: they yield a lot of false positives or negatives, potentially damage valuable DNA evidence, are not label-free, and can contaminate samples with chemical reagents (Bazyar, 2023). To overcome these challenges, μPADs can offer a compact size, portability, reduced risk of contamination, and safe sample storage for subsequent analysis (Bazyar, 2023; Harshey et al., 2023). Forensic investigations involve a wide array of diagnostic tasks, ranging from identifying and analyzing body fluids to detecting drugs of abuse and explosive residues. Common toxins detected include cyanide, ethanol, nitrite, and arsenic (Musile et al., 2023). They are also used to identify drugs and drugs of abuse, such as cathinones, 3,4-methylenedioxymethamphetamine (MDMA), xylazine, morphine, synthetic cannabinoids, benzodiazepines, cocaine, fentanyl, tetrahydrocannabinol, and ketamine (Musile et al., 2023). Moreover, µPADs are employed to detect psychoactive substances commonly used in drug-facilitated crimes, such as gamma-hydroxybutyric acid (GHB), metamizole, midazolam, flunitrazepam, and scopolamine (Musile et al., 2023). Additionally, μPADs can be employed for the preliminary analysis of gunshot residue, counterfeit pharmaceuticals, and evidence at crime scenes (Bazyar, 2023; Harshey et al., 2023; de Oliveira et al., 2017). While μPADs hold promise for security and forensic analysis, additional research is necessary to integrate them with portable and cost-effective platforms. This integration is crucial for ensuring that the developed sensors are both portable and quantifiable at the point of incident. Figure 6 shows the common applications of paper-based microfluidics in different sectors.

To improve the sensitivity and multiplexing capabilities of μPADs, researchers must focus on developing advanced microfluidic networks while incorporating signal amplification techniques. Interconnected designs with multiple channels and reaction zones enable the simultaneous detection of various targets, enhancing diagnostic efficiency. Incorporating signal amplification techniques, such as CRISPR/Cas biosensing systems combined with novel nanomaterials, can significantly boost detection sensitivity and signal strength, making μPADs more effective for complex analytical applications.

By leveraging artificial intelligence, machine learning, and the Internet of Things, data analysis and interpretation can become more efficient and accurate. Furthermore, the development of universal data processing applications compatible with various smartphone models would simplify result interpretation, making these platforms more user-friendly and widely applicable.

Optimizing patterning techniques can reduce batch-to-batch variations and enable stable mass production. Standardizing fabrication protocols and promoting the complete disclosure of methodologies in published works will enhance reproducibility, fostering greater confidence in μPAD-based technologies among researchers and users alike.

Automated test readouts are essential for improving detection reproducibility and minimizing user error. Integrating μPADs with portable detectors can enable real-time, on-site diagnostics and testing, making them invaluable tools in various real-world applications. These systems include smartphone-based detection systems, portable transmittance colorimeters, paired-emitter-detector diode (PEDD) systems, miniaturized electrochemical detectors, and automated lateral flow devices.

Achieving scalability in μPADs requires a focus on material consistency, user-centric development, and manufacturing precision. It is essential to use consistent materials for both prototyping and mass production, reducing the risk of costly redesigns. Additionally, prioritizing user needs is crucial by considering safety for non-professional use and designing devices with user-friendly dimensions that cater to the target audience. Finally, manufacturing precision must be assessed by evaluating parameters like volumes, setup costs, and batch sizes to ensure cost-effective and high-quality production of μPADs.

The primary scalability issues arise from the lack of field trials within the research. This gap is evident in many μPADs platforms, where researchers often neglect the importance of testing sensors with actual real-world samples. Consequently, there is a significant misuse of the term “proof-of-concept,” with published μPADs results often being difficult or impossible to reproduce. Many published works fail to properly mention the full protocols or describe them very poorly. To ensure μPADs perform effectively outside the laboratory, extensive field trials with real-world samples are critical. These trials help validate the performance of μPADs in diverse environmental conditions. Efforts should also focus on integrating sample collection, pretreatment, analysis, and data interpretation into single, user-friendly platforms tailored for resource-limited settings, ensuring practical usability.