Point-of-care (POC) diagnostics, which play an important role in personalized healthcare, have gained considerable attention owing to operations that enable medical staff to make rapid diagnoses and tailored treatment decisions. POC diagnostics offering affordable costs, easy operations without specially trained personnel, and rapid analytical consequences that have a positive correlation with the traditional clinical laboratory, are increasingly exploited in clinical diagnostic applications, especially if medical resources are limited [1,2,3,4]. Among the types of POC testing, paper-based analytical devices (PADs) have emerged as critical POC biosensors for disease monitoring and diagnostics, particularly in resource-constrained regions, for emergencies, and for in-house healthcare owing to their advantageous features, including simplicity, easy storage, disposability, and portability without relying on external devices [5,6,7,8]. There are three main classifications for PADs—lateral flow assays (LFAs), dipstick assays, and microfluidic PADs (µPADs) [9] with diversified signal readout approaches including colorimetry [10] luminescence [11,12] surface-enhanced Raman scattering (SERS) [13,14], photothermal methods [15,16], photoacoustic methods [17], and electrochemistry [18,19]. The most common detection approach in PADs is the colorimetric method, which can easily determine the presence of a target through color variations, for recognition even with the naked eye and without complicated instruments [20]. Despite the feasibility of PADs in POC diagnostics, comprehensive applications in diagnostic biosensors have still faced several challenges, such as low sensitivity and selectivity, poor quantitative discrimination, and limited stability [9,21], which have attracted research attention aimed at addressing these issues through signal-amplification methods.

Owing to its convenience and simplicity, the colorimetric lateral flow assay (CLFA) is one of the most prevalent diagnostic technologies for point-of-care applications, particularly for the detection of biomolecules [22,23,24], metal particulates [25,26,27], or food-contaminated pesticides [28,29,30]. In principle, colorimetric detection is associated with color development caused by an enzymatic or chemical reaction that can be seen with the naked eye or a semi-quantitative device. Nevertheless, it remains a great challenge to completely eliminate the background noise generated from the sample or the paper. Furthermore, due to the heterogeneity of the color distribution, evaluation of the final color is challenging. Such a limitation in sensitivity inhibits many critical applications, such as early detection of significant cancers and severe infectious diseases. With the rapid advancements in novel materials and nanotechnology, signal-amplification strategies that hold great potential to eliminate these limitations of CLFA have been developed in recent years. Significant effort has been dedicated to increasing light output yield, including controlling the physicochemical properties of the nanoparticle markers (such as metal label size and shape [31,32,33]), functionalizing nanocomposites with polymers [34,35], or employing enzyme-mimicking noble metal NPs [36,37,38,39].

Among the optical biosensors, luminescence-aided sensing, including chemiluminescence and bioluminescence, is a particularly compelling approach to signal transduction in many chemical sensing schemes due to the higher signal-to-noise ratio and the simplicity of the required measurement equipment. In general, detection with a luminescence-based device can be achieved by imaging or measuring the light emitted by bio-chemiluminescence or electro-generated chemiluminescence. Much effort has been dedicated to achieving an enhanced luminescence signal in PADs, mainly focusing on the choice of appropriate labeling agents, including the use of enzymes as labels in conjugation with enhancers to obtain improved signal intensity [73], metal-enhanced chemiluminescence [74], artificial pseudo-enzyme labels [75], and modified nanomaterial-based signal on-off mechanisms [76]. The use of nanomaterials has shown great promise in producing high-performance sensing devices due to their ability to act as the novel label of luminescent detection as well as being a platform to enhance the loading capacity of the luminescent labels. Liu’s group developed a novel dual-key-and-lock strategy-based ruthenium (II), or Ru(II), complex probe (Ru-FA) as an effective tool for formaldehyde detection in vitro and in vivo [77]. Ru-FA showed weak luminescence due to the photon-induced electron transfer (PET) process from the Ru(II) center to electron-withdrawing group 2,4-dinitrobenzene (DNB). Triggered by a specific reaction with formaldehyde (the first key) in an acidic environment (the second key), DNB is cleaved from Ru-FA, affording an emissive Ru(II) complex derivative, Ru-NR (Figure 2A). As a result of the PET process from the Ru(II) center to electron-withdrawing moiety DNB, Ru-FA itself displays weak luminescence, but its emission can be significantly increased after reacting with formaldehyde (key 1) in an acidic environment (key 2), accompanied by the production of emissive Ru-NR. This proposed system allows detection of formaldehyde at a 19.8 nM LOD, which is approximately 15 times higher than other paper-based analytical devices [78]. The on-off state of paper-based valves controlled by the rotation of paper discs is another strategy that has been used recently for improving a luminescence signal [79]. The rotational paper-based device was fabricated by assembling three designed paper discs using a hollow rivet. The on-off state of paper-based valves was easily controlled by rotation of the paper discs (Figure 2B). The integrated paper-based rotation valves can easily be controlled by rotating the paper discs manually, making it user-friendly for the untrained. In addition, the rotational valves are reusable, and the response time can be shortened to several seconds, which promotes the rotational paper-based device as offering great advantages in multi-step operations. Under the control of rotational valves, multi-step ECL immunoassays were conducted on a rotational device for multiplexed detection of carcinoembryonic antigen (CEA) and prostate specific antigen (PSA). The rotational device exhibited excellent analytical performance for CEA and PSA, which could be detected in linear ranges of 0.1–100 ng mL⁻¹ and 0.1–50 ng mL⁻¹ with limits as low as 0.07 ng mL⁻¹ and 0.03 ng mL⁻¹, respectively, which is approximately 10 times more sensitive than a paper-based fluorometric device [80]. Compared to other, conventional paper-based valves, the as-prepared platform could be dried for reuse, revealing its simplicity, rapidity, low cost, and excellent analytical performance. Due to its diverse structural polymorphism and an ability to switch on the signal upon binding to luminescence molecules, the non-canonical DNA secondary structure is another effective way to improve a luminescence signal. Sun et al. developed a paper-based µPAD based on a G-quadruplex-based luminescence switch-on assay for detection of the lead(II) ion (Pt²⁺) [81]. This type of suspended-droplet mode, paper-based µPAD uses wetting and gravity as a driving force. To fabricate the super-hydrophobic pattern on a paper device, a new microcontact printing-based method was applied by coating hydrophobic and transparent silicone polydimethylsiloxane (PDMS) on a glass slide attached with Teflon (a non-stick polymer allowing easy peel-off of the PDMS). For Pt²⁺ detection, G-quadruplex oligonucleotide and the iridium (III) probe, respectively, were added to the reaction zone and the detection zone of the test strip. The presence of Pt²⁺ allowed conformational change in the G-quadruplex, which can greatly enhance the luminescence emission of the iridium (III) probe (Figure 2C). The proposed platform was integrated into an inexpensive, battery-powered compact device for routine portable detection using a smartphone. Pt²⁺ was detected at low concentrations within the linear range from 10 nM to 100 nM. Excited-state proton transfer (ESPT) with huge luminescence Stoke shifts and an ultrafast response is another way to enhance the luminescence signal intensity and has generated great interest. Recently, an ESPT concept-based luminescence sensor was designed for discriminative detection via enol-keto tautomerism [82]. To improve the sensitivity, two-dimensional (2D) nanosheets of a metal-organic framework (MOF), Cd₂(2,5-tpt)(4,5-idc)(H₂O)₄, were synthesized via top-down liquid ultrasonic exfoliation technology for sensing water in dimethylformamide (Figure 2D). This sensor can serve as a dual-sensing mechanism along with luminescence color change via shifted emission (green to yellow) in low water content and can be a turn-off method in high water content. Such a 2D nanosheet-sensing platform was applied to a paper test strip for ease of water detection with a rapid response (<30 s), long-term stability, pH stability, good reusability, high selectivity, broad-range detection (0–50% v/v), and a low LOD value (0.25% v/v) that is considerably lower than the conventional MOF-based sensing platform [83]. The recent improvements in materials and nanotechnologies have created new opportunities to further enhance luminescence signals through numerous strategies, including the use of proper labeling agents, a choice of fabrication process, or the use of an electrical energy supply. These approaches have shown great promise in producing handheld, paper-based devices with more sensitive detection of analytes using miniaturized devices. With a comprehensive understanding of the luminescent PAD, new generations of these sensing systems promise to solve the limitations of previously established devices, and could be employed in commercial applications.

SERS is a sensing technique that generates and amplifies inelastic light scattering of molecules when they are adsorbed on metals like gold, silver, and copper. This scattering signal could be enhanced (up to 10 orders of magnitude) by modulating the frequency of excitation light and the localized surface plasmon resonance (LSPR) of the metallic nanomaterials, relevant to the Stoke and/or anti-Stoke lines of molecules. In a SERS-based assay, specific tags made of nanostructures, and molecules with known Raman fingerprints, are the detection agents. Measuring the peak intensity of Raman molecules allows for quantification of particle and analyte concentrations. Consequently, SERS has become a powerful tool in diagnostics due to its rapid, sensitive, and multiplexed outcomes.

Manipulating the shape, size, and composition of the nanostructure has shown great promise in improving the SERS signal. In particular, the gold nanostar (GNS) providing high SERS performance was used as a Raman reporter for PAD fabrication that showed significant signal intensity in the detection of bisphenol A (BPA) [84]. Gold nanostars with a sharp branched shape are great material for tuning plasmonic properties, providing the strongest SERS activities. GNSs were used as a SERS nanotag in comparison with spherical AuNPs. Both GNSs and AuNPs were tagged with the Raman reporter molecule ATP, followed by incubation with an anti-BPA antibody to form GNS/AuNP-antibody-ATP conjugates. BSA was subsequently used as a blocking agent, and 4,4-bis(4-hydroxyphenyl) valeric acid (BHPVA) was allowed to be bound by BSA to prepare the BHPVA-BSA conjugate. An LFA strip was used, consisting of a nitrocellulose membrane, a conjugate pad, an absorbent pad, a sample pad, and a support sheet. On the conjugated pad, the GNS/AuNP-ATP-antibody conjugate was dispersed, and the nitrocellulose membrane was coated with BHPVA-BSA and a goat anti-rabbit antibody as a control. For BPA detection, the BPA solution was dropped on the sample pad, where the analyte migrates to the membrane and conjugate pad, and finally moves to the absorbent pad. The color change in correlation with BPA concentration caused by the presence of GNS allowed quantitative detection of BPA (Figure 3A). The as-prepared SERS-LFA allows BPA detection at concentrations ranging from 0.05–60 ppt with a detection limit down to 0.073 ppt, indicating it is about 136 times more sensitive than the PEGylated AuNP-based LFA [85]. An alternative strategy for SERS enhancement is multilayer nanostructure fabrication that takes advantage of all employed compositions. As such, a SERS paper-based sensor was constructed by decorating paper chips with a sandwich structure of 3D silver dendrite (SD)/electropolymerization of molecular identifier (EMI)/silver nanoparticle (AgNP) (Figure 3B) [86]. The SD was synthesized on the surface of paper chips using a chemical reduction growth technique followed by immobilization of the EMI layer that serves as a specific recognizer. Subsequently, the core enhancement AgNP layer was integrated on top of the sensing chip. The as-prepared SERS paper chip was successfully applied for in situ detection of imidacloprid (IMI), showing ultra-high sensitivity with a detection limit of 0.02811 ng mL⁻¹, 2000 times more sensitive than other paper-based organic–inorganic manganese (II) halide hybrid sensors [87]. This multiple SERS enhancement paper chip holds great potential for screening a variety of contaminants. The plasmonic alloy of different noble metals is another effective way of providing high-quality opportunities for tuning plasmon resonance. Taking advantage of the hotspot effect that creates an intense electromagnetic field near the plasmonic structure, the plasmonic alloy has been used for sensitive biosensor techniques such as SERS. Recently, a paper-based plasmonic substrate with a plasmonic alloy of Au/Ag nanocomposites on hierarchical cellulose micro-/nanofiber matrices for highly sensitive metal-enhanced fluorescence (MEF) and SERS biosensor applications was developed [88]. In the fabrication process, Au and Ag were deposited on the cellulose fibers at a low-temperature wafer level below 100 °C to avoid damage to the paper substrate and cellulose fibers (Figure 3C). The Au/Ag nanocomposite was thermally evaporated from the surface of the cellulose fiber. The integration of Au/Ag nanocomposites that act as a plasmonic alloy allows two distinct extinction peaks from Au and Ag to combine into a single peak. This paper-based plasmonic alloy substrate enables a two-fold increase in fluorescence signals and selective MEF signals, compared to Whatman chromatography paper. The proposed sensing device allows detection of folic acid at a picomolar level (LOD 1 pM), which is 1000 times more sensitive than other carbon dot-based paper devices (0.28 µmol/L) [89]. Paper-based devices have also been fabricated for the detection of foodborne bacteria. However, the low intensity of conventional Raman spectroscopy and fluorescence interference makes it difficult to detect biological samples in the complex. One strategy to deal with this challenge is to develop three-dimensional (3D) SERS substrates that offer a larger surface area for absorbing more probe molecules, and that generate more hotspots for analyte binding. Such an idea was applied to developing a novel, label-free, filter paper-based 3D-SERS detection platform to detect and classify common foodborne bacteria, including E. coli, Listeria monocytogenes, and Staphylococcus aureus [90]. Black phosphorus-Au (BP-Au) nanosheets were used as a SERS substrate that could generate abundant hot spots, showing great potential to serve as excellent SERS substrates (Figure 3D). The BP-Au sheets were prepared by conjugating AuNPs on prepared BP sheets to form BP-Au sheets. Subsequently, the BP-Au sheets were applied to the filter paper surface. This BP-Au filter paper-based 3D-SERS substrate exhibited remarkably improved Raman signals, allowing for specific recognition and classification of three types of target bacteria at concentrations as low as 10⁻⁷ CFU/mL, with an enhancement factor of 2.4 × 10⁴ (17 times higher than the Au/Ag paper substrate) [91]. This paper-based sensing device would be beneficial in practical applications for food safety. The employment of enzyme-like noble metal NPs as substrates for SERS is another approach that significantly improves SERS signals. Taking advantage of nanozymes for the catalytic degradation of methyl mercury, several paper-based SERS sensing devices using nanozymes have been developed. In particular, Liu et al. reported fabrication of Au-NiFe-layered double hydroxide (LDH)/rGO nanocomposites, which are not only efficient nanozymes with oxidase-like activity but also efficient SERS substrates to determine organic mercury [92]. Under the free radicals of electron paramagnetic resonance (EPR) spectra and the binding energy of an X-ray photoelectron spectrometer (XPS), upon the oxidase-like catalytic reaction, the oxygen molecules are captured, leading to increased O₂ radicals, while MeHg is degraded, resulting in the release of CH₃ radicals. The as-prepared Au-NiFe LDH/rGO nanocomposite could be used to effectively degrade and remove 99.9% of organic mercury in two h without secondary pollution of Hg²⁺, and a low concentration of MeHg was detected (as low as LOD 1 × 10⁻⁸ M), which is 10 times more sensitive than the conventional paper-based device [93]. To date, much effort has been devoted to enhancing the signal output for SERS-based PADs. The innovative achievements in the bioscience and nanomaterial science fields hold great promise for boosting fabrication of highly sensitive, low-cost, and miniaturized devices for point-of-care applications.

The photothermal activity of nanomaterials is another exciting property that has been exploited in recent years for the enhancement of paper-based sensing assays. Generally, the application of photothermal agents in the field of bioanalysis is based on light-to-heat conversion, which can be significantly intensified by using appropriate photothermal agents and a proper recognition system. Consequently, photothermal agent-mediated detection of a target can be carried out using a thermometer as a readout where the temperature signal is linear in relation to the target concentration. A wide variety of materials, including AuNPs [94], carbon NPs [95], quantum dots [96], and plasmonic NPs [97], have been exploited as photothermal agents for broad application in various fields. AuNPs that exert excellent photothermal signals have been extensively investigated to improve sensitivity [98,99]. Recently, a dual-signal biosensor based on multifunctional MnO₂-Au was developed, enabling rapid achievement of qualitative information (visible to the naked eye) and quantitative photothermal data when detecting furazolidone antibiotic residues [100]. The fabrication process includes functionalization of AuNPs with MnO₂ nanoflowers to form MnO₂-Au signal probes that exhibit high colorimetric/photothermal properties. The MnO₂-Au probe is then conjugated with a specific antibody (anti-CPAOZ mAb), followed by incubation with BSA to block non-specific binding. MnO₂-Au exhibits a remarkably enhanced light-to-heat conversion effect attributed to the MnO₂ nanoflowers’ high capacity to carry AuNPs. The flow immunoassay strip was composited from a nitrocellulose membrane, an absorbent pad, a sample pad, and a conjugate pad. Consequently, AOZ (the metabolite of FZD) can be quantitatively measured on the basis of both color change and heat signal (Figure 4A). The as-prepared immunoassay allows sensitive and comprehensive detection of FZD in food at low levels, attributed to the excellent light-to-heat conversion capacity of the MnO₂-Au signal probe. FZD was detected at LOD 1 ng mL⁻¹ and 0.43 ng mL⁻¹ by colorimetric signal measurement and photothermal signal, respectively, indicating eight-fold improved sensitivity compared to the conventional colorimetric, gold-based lateral flow immunoassay [101]. The excellent performance exerted by this sensing device would be a promising sensing platform for broader application in point-of-care settings.

Recently, numerous advances in microfabrication techniques have been made, offering more facile and sensitive devices that possess many advantages over the conventional approaches. The microfluidic platform-based photothermal sensing assay is one of the currently emerging systems that hold great potential for paper-based point-of-care testing [102,105,106]. Microfluidic technologies have become increasingly powerful tools for developing point-of-care diagnostics because of their distinct advantages. However, the conventional microfluidic devices fabricated on silicon or glass surfaces require intricate design processes with complex instruments, limiting their practical application [107]. On the other hand, paper-based microfluidic devices exhibit great properties, including low cost, simplicity, sensitivity, and portability, making them a promising tool for point-of-care testing. The integration of the microfluidic platform with an intelligent sensing element is a novel strategy to compensate for the limitations of conventional microfluidic devices through synergistic performance in a single pattern. A photothermal microfluidic pumping approach was developed for an on-chip µPAD in a spatiotemporally controllable and contactless manner [102]. A multiplexed cargo reservoir fabricated from thermo-responsive poly(N-isopropylacrylamide)-acrylamide hydrogel-doped graphene oxide (GO) was immobilized on a paper surface (Figure 4B). The integration of composite hydrogels on highly photothermal-capable GO resulted in an on-chip phase transition of the composite hydrogels in a switch-like fashion. As a consequence, a strong pumping dynamic of the cargos from hydrogel to the reaction zones was achieved. By remotely controlling the laser power, GO density, and irritation time, the microfluidic pumping performance could be spatiotemporally manipulated. To demonstrate the efficiency of this pumping strategy, horseradish peroxidase and FeCl₃ were used as the model cargoes to immobilize 3,3′,5,5′-tetramethylbenzidine and Prussian blue, respectively, as photothermal probes for µPAD-based colorimetric reactions. Owing to its high integrability in lab-on-chip paper-based devices, its controllable spatiotemporal capacity, and its features of being contactless and highly flexible, this novel microfluidic pumping platform shows great potential for numerous microfluidic applications.

Nevertheless, without the assistance of biological materials, photothermal sensing devices show remarkable limitations when applied to bioanalysis, including immunoassays. To overcome this challenge, Fu et al. developed a photothermal biosensing approach by integrating a photothermally responsive poly methyl methacrylate (PMMA)/paper hybrid disk (PT-Disk) in a clip magazine-assembled fashion [103]. The clip units consist of a honeycomb-patterned paper substrate and a hydrogel carrier fabricated from a PMMA cellulose cover conjugated with a specific antibody. These clip units were rotationally assembled on a magazine bearer composed of a strip-formed, channeled-paper sheet and an upper PMMA cover. To accelerate the photothermal conversion efficiency, Fe₃O₄ was captured on the paper substrate with an antibody and transported to a dual-functional probe—Prussian blue (PB) NPs that exert both colorimetric and photothermal activities. Subsequently, the dye-enveloped thermo-responsive hydrogels were loaded in clip wells. Upon laser irradiation, the photothermal effect of the PB NPs stimulated a rise in the dye solution’s temperature in a dose-dependent fashion, leading to the release of dye solution from the clip units to the surrounding magazine-bearing channels, which could be quantified by on-chip rulers and a portable camera (Figure 4C). The as-proposed sensing device allows analyte detection in a tri-mode signal output approach, including colorimetric readout on the paper disk for thermal-image and distance-reliable visual quantification. Using this novel sensing platform, the cancer biomarker (a prostate-specific antigen) was detected at LOD 1.4–2.8 ng mL⁻¹, which was lower than the previously reported practical diagnostic threshold [108,109].

Due to their unique properties, photothermal-based devices have been employed for broad application in various fields, including controlling evaporation rates under solar irradiation. The hydrophobic surface is well-known for the design of water treatment systems. However, interfacial thermal resistance events occurring after hydrophobic treatment usually lead to remarkably low energy efficiency, limiting the water treatment process. To address this limitation, a carbon nanotube (CNT)-modified superhydrophobic photothermal membrane was recently fabricated on a filter paper substrate for effective water desalination upon solar irradiation [104]. In particular, the superhydrophobic photothermal membrane was composited from the CNT and polyvinylpyrrolidone (PVP) modified with 1H,1H,2H,2H-perfluorodecyl (CNT@PVP). The CNT@PVP composite was sprayed on a filter paper surface to produce a photothermal membrane that is impermeable to water. The membrane was placed onto an expanded polystyrene-prepared bottom stand for flexible floating and heat localization (Figure 4D). The as-prepared device exhibited high energy efficiency (91%) with an increased evaporation rate of 1.41 kg m⁻² h⁻¹, indicating higher desalination efficiency than previously established devices [110,111]. The key advantages of the photothermal conversion system include a high light-to-heat capability, reusability, stability, low cost, and portability, making it a promising tool for signal amplification and quantification of PADs.

Photoacoustic (PA)-enhanced signals have been developed in a few PAD-based biosensing platforms for cryptococcal antigen [112], heparin [113], and ALP [114]. Photon energy, strongly absorbed by chromophores, is converted into mechanical energy via thermal expansion, resulting in the production of acoustic waves as PA signals [115,116]. Zhao et al. developed photoacoustic-based LFA using AuNPs to quantitatively measure cryptococcal antigen (Figure 5A). Compared to semi-quantitative colorimetric analysis, the PA detection method improves the sensitivity of LFAs owing to the strong LSPR of AuNPs and the effective elimination of a PA background signal associated with ambient light noise. Light absorption was specifically designed to enhance the PA signal with effective minimization of the background signal from paper substrate. To confirm the enhanced effect of the PA method on LFA, the limit on detections measuring cryptococcal antigen was calculated for three different measurement methods, including colorimetric measurements (1.1 ng/mL), chop mode PA detection (0.57 ng/mL), and scan mode PA detection (0.010 ng/mL), indicating the capability of the PA method to decrease LOD by more than 100 times. This PA-based LFA offers advantages in terms of high sensitivity, reduced system noise, strong and reliable PA signal generation from AuNPs, and minimization of expensive photodetectors and optical filters [112]. Another paper-based photoacoustic sensor for direct detection of heparin was fabricated for point-of-care measurement of heparin activity in human blood samples via fingerprick-sized blood samples (Figure 5B). A cellulose-based photoacoustic heparin sensor was fabricated by loading cationic dye (Nile blue A) onto polyethylene glycol (PEG)-modified Whatman filter paper. Heparin, a negative-charged polysulfated glycosaminoglycan, can interact with Nile blue A to exhibit PA spectral alternation. This PA-based sensor showed LOD at 0.28 U/mL of heparin in human plasma with a turnaround time of 3 min and at 0.29 U/mL in whole blood within 6 min. The correlation of heparin, turnaround time, and sample requirement is comparable to an activated clotting time test. This PA sensor for heparin can monitor not only heparin concentration but also heparin activity in human serum samples, which is exploited as an affordable and disposable heparin sensor [113]. Instead of AuNPs, Zhang et al. developed a portable photoacoustic device for determination of ALP in serum using silver nanoparticles with excellent photo-stability and reproducibility (Figure 5C). The breakdown of sodium L-ascorbyl-2-phosphate into ascorbic acid was catalyzed by the ALP enzyme, thereby converting AgNO3 into AgNPs. Under irradiation from a modulated 638 nm laser, a strong PA signal generated by AgNPs due to their localized plasmon resonance was detected by the portable PA device. After optimizing the reaction condition, this PA-based sensor could detect ALP in a concentration range of 5–70 U/L with LOD of 1.1 U/L. This rapid portable PA device exhibits favorable reliability and stability for determination of ALP in human serum with high sensitivity, providing a PA-based analytical strategy to detect disease-correlated biomarkers [114]. Although the reproducibility of PA-based PADs should be thoroughly validated due to the intrinsic point-scanning reading of the PA technique, the PA technique may exhibit promising potential to measure biomarkers in clinical realities.

Photoelectrochemical (PEC) detection has drawn increasing attention due to its promising analytical features, including low cost, simple equipment, portability, persuasive selectivity and sensitivity, and low background, facilitating high-throughput and quick assays [117,118,119,120]. Driven by these advantages, paper-based PEC systems that integrate the inherited excellence of cellulose paper and PEC bioanalysis have been fabricated [117]. The PEC platform stems from the electrochemical ideal, which can convert light energy into chemical and electrical energy, hence ameliorating sensitive detection through the modification of photocurrent responses into photoelectrochemical reactions based on analytes and specific photoactive matrix/probe interactions [117,121,122]. Photoactive electrons can be stimulated upon excitation by light, and conveyed to an electrode to generate a vigorous photocurrent signal, for which a signal-off or signal-on readout method is employed [117,122].

Despite outstanding achievements that have been obtained in lab-on-paper analytical devices, certain challenges for employed electrodes in assisting paper-based devices still exist. First, the most serious reason is associated with conductive inks that only cover the paper’s surface and are not assembled into the interior structures, leading to limited conductivity and, eventually, considerable influence on the sensitivity of the devices. The next problem is associated with practical stability under harsh conditions due to too-weak interactions between cellulose fibers and conductive materials. Finally, highly efficient lab-on-paper analytical devices urgently require remarkable capacity to generate, transmit, and measure electrical signals [123]. Therefore, PEC-based signal amplification enhancement that can address these issues is necessary in order to minimize the number of assays, but still reach high sensitivity with a very low concentration. In the last few years, diverse strategies have been proposed to amplify photoactive signals for PEC-sensing fabrication, most of which have been employed in electron acceptor/donor regulation [120,123,124,125,126], photoactive materials, nanozymes [119,120,127,128,129,130,131,132,133,134,135], or light sources [136,137,138,139,140,141,142]. Among them, functional nanomaterial integration is an emerging potent route to reach giant, efficient lab-on-paper analytical devices, and diverse photoactive nanomaterials have been immobilized on cellulose fibers. Although it is well known that an excited light source is requisite for a photoelectrochemical platform, physical light source-related instruments are also required [142]. Therefore, to achieve instrument miniaturization as well as operational simplification, luminol-based chemiluminescence emission was exploited as an interior light source, which offers multitudinous advantages, including low cost, simplicity, high sensitivity, and a rapid response [136]. Lan et al. designed and constructed a novel 3D-rGO/cellulose-based photoelectrical lab-on-paper analytical device to determine thrombin (TRB) in serum samples, a procedure typically utilized for Alzheimer disease and cardiovascular disease diagnosis [142]. The combination of reduced graphene oxide and Au flower in paper cellulose fiber facilitates superior conductivity as well as biocompatibility. ZnO anchored/nitrogen-doped carbon dots are immobilized in this system and stimulated by strong chemiluminescence of a luminol–H₂O₂ system to generate a photocurrent signal (Figure 6A). This proposed sensing performed with excellent sensitivity, as well as specificity in thrombin determination, and can detect very low concentrations of 16.7 fM.

Alternatively, sensing can obtain the dual functions of target recognition and signal transduction, which are widely employed in cyto-assays due to their triggering of the signal of target recognition [123]. Recently, the electron acceptor/donor methodology has been widely investigated for enzyme-catalyzed consumption/generation, which are exerted as co-reactants in hole-oxidization reactions and electron moderation, leading to cathodic/anodic photocurrent signal production [143]. Li et al. fabricated a novel sensing stand on the typical catalytic reaction of a hemin/platinum nanoparticle (Pt NP) trunk-branching-decorated DNA dendrimer toward H₂O₂ [143]. Pt NPs have been widely exerted in bioassays due to their excellent inherited properties involving small size, diverse enzyme-mimic activities, and especially catalytic activity toward H₂O₂ for electron acceptor (O₂) production [145,146]. Thus, in this proposed sensor, the DNA dendrimer serves as enzyme-like activity enhancement and synergy catalysis with Pt NPs, as well as hemin, to catalyze H₂O₂ and increase electron acceptors; hence, it can determine the target miRNA-141 over a wide range, from 0.5 fM to 5 nM with a limit of detection at 0.17 fM (Figure 6B).

In photoactive material-associated PEC paper-based devices, photoactive species play an essential role serving as a light absorber [119]. In particular, emerging nanozymes are being intensively investigated as an expected alternative to natural enzymes due to their many intrinsic qualities, including superior stability, enzyme-like capabilities, and wide pH operating range [128]. For instance, Li and coworkers fabricated photoelectrochemical paper-based sensing to detect carcinoembryonic antigen (CEA) based on the modification of paper with N-carbon dots/TiO₂–Pt in a seed-mediated growth method [144]. First, TiO₂ seeds were prepared from a TiCl₃ solution in a mixture maintained at 80 °C for 2 h and then dried at 450 °C for 2 h. In the next step, these TiO₂ seeds were spread onto cellulose fiber (Pt/PWE) and equilibrated at 37 °C, repeating the process three times. The proposed TiO₂ NP-modified photoelectrochemical lab-on-paper analytical devices can determine CEA concentrations based on the reduction of the photoelectrochemical signal, because they bind with CEA at the MCF-7 cell surfaces in human serum (Figure 6C). This novel platform has good biocompatibility, is portable, and has eco-friendly devices that can detect low levels in limitations of 1.0 pg mL^–1 over a broad linear range of 0.002–200 ng mL^–1. Additionally, Li and colleagues also employed this strategy to develop an ultrasensitive PEC paper-based device to determine miRNA-141 from a two-enzyme-engineered DNA walker and a TiO₂/CeO₂ heterojunction (Figure 6D) [128]. During cancer development, miRNA-141 is expressed in large proportions, and thus, acts as a potential marker for cancer diagnostics and assessment. In the presence of miRNA-141, the protecting probes would be kept away, leading to activated walker probes, creating an endonuclease cleavage reaction, and eventually, retrieving a signal output. The novel sensor can verify miRNA-141 in real human serum with LOD at 0.6 fM, and can ensure long-term stability and highly satisfactory selectivity.

Aptamer-integrated PADs are emerging as potential diagnostic sensors due to the outstanding aptamer advantages of more reasonable costs, high thermo-stability, and non-toxicity, as well as fewer batch-to-batch variations in comparison to antibodies [147,148]. A paper-based nucleic acid amplification test has been developed with the support of nucleic acid amplification and clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas) systems to improve the sensitivity of PADs.