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LAMP is a nucleic acid amplification technique first introduced by Notomi et al. in 2000 [21]. LAMP employs four to six primers that specifically recognize distinct regions of a target gene, enabling highly selective amplification. The LAMP reaction proceeds in two main stages: the formation of a dumbbell-like structure and subsequent cyclic amplification (Fig. 1).

Fig. 1. 
Schematic of the LAMP principle. The reaction consists of (1) dumbbell-shaped DNA structure formation, and (2) cyclic amplification through repeated primer binding and elongation under isothermal conditions. The suffix “c” in sequence labels (e.g., F1c, B2c) indicates the complementary strand.

In the initial stage, the strong strand displacement activity of the Bst polymerase allows the separation of double-stranded DNA into single strands without the need for thermal denaturation. The front inner primer (FIP) binds to the upstream region of the single-stranded DNA at the 3' end and initiates polymerization. Simultaneously, the front outer primer (F3) binds downstream of the FIP-binding site, generating a new double-stranded region. This binding displaces the FIP-initiated strand by a strand-displacement mechanism. The displaced strand is designed so that its 5' region contains a sequence complementary to part of the FIP, allowing it to form a self-hybridized loop structure. The backward inner and outer primers (BIP and B3) function similarly, resulting in the formation of a characteristic dumbbell-shaped DNA structure.

In the amplification stage, the dumbbell structure, with its open 3' end, repeatedly opens and reforms loops during synthesis. These loop regions continuously expose primer-binding sites, facilitating exponential amplification. The inclusion of loop primers (LF and LB) further enhances efficiency by providing additional initiation points within the loop regions. Together with the strand displacement activity of the Bst polymerase, this architecture drives a rapid and continuous amplification process that exponentially increases the target DNA concentration.

LAMP achieves high sequence specificity using four to six primers that collectively recognize six to eight distinct sites on the target gene. Single-base mismatches can inhibit amplification, thereby ensuring high fidelity. Furthermore, its unique loop-driven, non-cyclic amplification mechanism enables the rapid accumulation of amplified products, allowing for the detection of extremely low levels of nucleic acid targets. These structural and functional features make LAMP a powerful molecular diagnostic technique capable of high-sensitivity analysis without the need for complex thermal cycling equipment.

LAMP differs markedly from conventional PCR in several aspects, including diagnostic principles, reaction speed, equipment requirements, and analytical methods (Table 1). In this section, we compare these two technologies from the perspective of their applicability to on-site platforms.

PCR relies on the cyclic repetition of three steps (denaturation, annealing, and extension), each of which requires distinct temperature conditions (approximately 95, 40–60, and 72°C, respectively) [22]. This process enables the amplification of trace amounts of the target nucleic acids within approximately 2 h, thereby providing relatively high sensitivity and specificity. Nevertheless, PCR exhibits limitations in terms of the reliable detection of targets present at extremely low concentrations. Although digital droplet PCR (ddPCR) allows the detection of ultralow concentrations, it is still challenging to implement outside laboratory environments [23]. Moreover, both the PCR and ddPCR platforms require stable power supplies and rely on bulky, high-cost instrumentation, which makes portable or on-site operations highly impractical. Consequently, PCR-based diagnostics must be performed in centralized laboratories and typically require at least 12 h for sample transportation, preprocessing, and result reporting (Fig. 2) [24].

Fig. 2. 
Schematic illustration of virus detection workflows using PCR, conventional LAMP, and on-site LAMP platforms. The figure compares the sample delivery, amplification, and result reporting steps across the three approaches. Both PCR and conventional LAMP require centralized laboratory processing, resulting in longer turnaround times of 12 h or more for PCR and at least 6 h for conventional LAMP. In contrast, on-site LAMP platforms based on paper, microfluidic, or hydrogel enable decentralized testing and significantly reduce the overall detection time to approximately 50 min.

In contrast, LAMP operates at a constant temperature of 60–65°C and does not require thermal cycling. The reaction can be performed using a simple heating device, making LAMP far less dependent on specialized instrumentation. This feature reduces the total diagnostic time to 30–50 min. Unlike PCR, which uses two primers (forward and reverse), LAMP employs four to six primers that recognize multiple distinct regions within the target gene. This multisite recognition reduces the likelihood of nonspecific amplification and contributes to its high specificity [21].

The two methods also differ significantly in terms of amplification yield and detection modality. PCR typically achieves exponential amplification, with product quantities doubling in each cycle, and requires specialized analytical tools such as fluorescent probes or electrophoresis for detection. Therefore, it is well-suited for quantitative analysis. In contrast, LAMP generates large amounts of DNA through a cyclic amplification mechanism within a short timeframe. During the reaction, pyrophosphate is produced as a byproduct and reacts with magnesium ions (Mg²⁺) to form a visible white precipitate of magnesium pyrophosphate (Mg₂P₂O₇), allowing for a direct visual readout [25]. In addition to turbidity-based detection, various colorimetric and fluorescence-based approaches utilize changes in pH, Mg²⁺ concentration, or dye interactions [26-29]. These intuitive readout methods are optimal for qualitative analysis and are particularly advantageous in POCT scenarios where access to analytical equipment may be limited.

Despite these advantages, conventional LAMP technologies have several limitations that hinder their direct application in the field. In current workflows, sample preprocessing is typically performed separately from amplification, and often requires additional equipment and trained personnel. This separation not only increases the overall turnaround time but also increases the risk of sample degradation or contamination during transport to centralized laboratories. Consequently, although faster than PCR, conventional LAMP-based diagnostics still require several hours, including sample handling, transport, and preprocessing, which typically take up to six hours of total diagnostic time (Fig. 2) [30].

To overcome these limitations, portable LAMP systems that integrate sample preparation, amplification, and interpretation of results on a single platform have been developed. Recent efforts have focused on implementing LAMP on platforms such as paper, microfluidic systems, and hydrogels. These platforms aim to streamline the entire diagnostic workflow for use outside laboratory settings, offering a significant time advantage by delivering results in less than 50 min compared to the hours required by conventional methods (Fig. 2) [31,32]. On-site LAMP technologies are currently being actively explored as viable alternatives to address the practical constraints of conventional LAMP, bringing the method closer to true field-ready diagnostics.

Paper has gained significant attention as a substrate for POCT platforms because of its light weight, low cost, ease of disposal, and environmental friendliness compared to other materials [33-36]. In particular, its ability to store reagents in a dried, immobilized form facilitates long-term storage and field deployment, reducing the dependence on user expertise during operation [37]. These advantages have led to extensive research on paper-based on-site and POCT platforms, most notably, the lateral flow assay (LFA), which has been commercialized for the self-diagnosis of COVID-19 [38-40].

However, conventional LFA systems have limited sensitivity, which makes it difficult to detect viruses during the early stages of infection [41]. To address this limitation, several strategies have been proposed that combine LAMP with LFA-based signal readout [42-45]. For example, Witkowska McConnell et al. developed a system in which the LAMP primers FLP and BLP were labeled with biotin and FITC, respectively (Fig. 3 (a)) [42]. The resulting LAMP amplicons were captured using antibodies and visually detected using an LFA strip. Batule et al. demonstrated a method in which the amplified product was eluted and detected using a double-stranded DNA-binding LFA structure, enabling visual interpretation of the results [43]. Seok et al. further advanced this approach, integrating the LAMP and LFA steps into a fully unified one-step platform, eliminating the need for separate processes [44].

Fig. 3. 
Representative examples of the paper-based LAMP platform. (a) Schematic of LFA-LAMP endpoint detection with dual-labeled primers for visual readout. Adapted from Ref. [42]. (b) Multiplexed paper device for wastewater SARS-CoV-2 detection using CRISPR-Cas12a and RT-LAMP. Adapted from Ref. [47].

In addition, several systems have been developed in which the LAMP reaction is performed directly on paper substrates, with detection achieved using colorimetric or fluorescence-based signals within a single device [46-49]. Nguyen et al. designed a rotary paper-based device combining RT-LAMP with the food dye carmoisine [46]. This system allows multiplexed visual detection of SARS-CoV-2 and other pathogens through color changes in compartmentalized vertical chambers, making it suitable for use in low-resource field settings. These devices integrate reaction and detection zones into a single paper structure, enabling simplified fabrication, reduced manufacturing costs, and streamlined workflows from sample loading to result interpretation.

Taking the approach further, Cao et al. introduced a CRISPR-Cas12a system into a paper-based LAMP platform to address the issue of nonspecific amplification (Fig. 3 (b)) [47]. Their device detected SARS-CoV-2 N, E, and S genes in wastewater samples, with a limit of detection ranging from 0 to 310 copies/mL. The system demonstrated a sensitivity of 97.7% and semiquantitative accuracy of 82%. Moreover, the entire process, from sample collection to interpretation, was completed within 2 h without the need for complex equipment. The inclusion of Cas12a enabled the specific recognition of the amplified target sequence, thereby reducing false positives and improving both sensitivity and specificity. This platform is a strong example of a fully integrated field-deployable diagnostic system that combines practicality and broad applicability to various pathogens.

Microfluidic diagnostic platforms, which utilize microfluidic channels to perform precise biochemical reactions with small sample volumes, have been widely applied across various biological and analytical fields [50-52]. These systems are particularly well-suited for integration with LAMP technology, because they enable precise control over key processes such as reagent mixing, droplet generation, sample concentration, and temperature regulation. Given the complexity of LAMP, which involves multiple reagents and requires isothermal amplification under tightly controlled conditions, microfluidic platforms offer structural advantages for achieving consistent reaction performance.

Microfluidic chips not only provide accurate thermal and fluidic control, but also enable the integration of the entire diagnostic workflow, including sample preprocessing, amplification, and signal readout, within a single device. This feature allows for the development of fully automated diagnostic systems that follow a sample-in, answer-out format. Additionally, microfluidic platforms can be readily adapted for multiplexing by modifying the channel designs, allowing for the simultaneous detection of multiple targets. For example, Suarez et al. developed a LAMP-based microfluidic chip capable of detecting Influenza A, Influenza B, and SARS-CoV-2 simultaneously (Fig. 4 (a)) [53]. By sharing temperature regulation and fluorescence detection modules across channels, the system achieved limits of detection of 89 RNA copies for Influenza A, 245 for Influenza B, and 38 for SARS-CoV-2, with analysis times of less than 48 min and 100% specificity.

Fig. 4. 
Representative examples of the microfluidic-based LAMP platform. (a) Rotary microfluidic disc integrating chambered LAMP detection for respiratory viruses including SARS-CoV-2 and influenza. Adapted from Ref. [53]. (b) Digital droplet LAMP system enabling semi-quantitative multiplex detection and classification of targets based on precipitate intensity. Adapted from Ref. [57].

Other studies have reported similar results. Xie et al. integrated a colorimetric LAMP reaction into a digital microfluidic system, enabling visual signal readout on a smartphone for simple, field-friendly diagnostics [54]. Zhou et al. developed a microfluidic LAMP chip for the detection of porcine coronaviruses, utilizing a CD-shaped device design that allowed multiplexed analysis of multiple pathogens [55].

Recently, droplet microfluidics has been employed to implement digital LAMP [56,57]. Jin et al. proposed the StratoLAMP platform, which compartmentalizes individual LAMP reactions into hundreds of thousands of microdroplets, and enables dual-target quantification through visual signal stratification based on Mg₂P₂O₇ precipitation (Fig. 4 (b)) [57]. The system achieved 94.3% accuracy using a deep-learning algorithm for object recognition and provided label-free quantification without relying on fluorescence. By adjusting the primer concentrations, the amount of precipitate in each droplet could be modulated, enabling the simultaneous quantification of two different targets. This approach shows great promise for on-site virus detection, which requires high sensitivity, high precision, and label-free detection.

However, despite these advantages, microfluidic LAMP systems have several limitations. The fabrication of microfluidic chips typically requires high-precision manufacturing techniques that increase the production complexity and cost relative to paper-based platforms. In many cases, external components such as pumps, valves, and thermal controllers are required, which can hinder portability and ease of use in on-site virus detection. To enhance the practicality of microfluidic LAMP systems for on-site diagnostics, further development is required to simplify the device architecture and enable cost-effective fabrication of disposable components [58].

Hydrogels are composed of three-dimensional polymer networks containing large amounts of water and are widely used in various biological analysis systems because of their high water retention capacity, diffusion control ability, and biocompatibility [59-61]. The semi-solid nature of hydrogels provides mechanical resilience and chemical stability, allowing them to maintain their reaction components even under temperature fluctuations and pH changes during amplification. These properties enable the stabilization of biomolecules, such as enzymes and primers, for extended periods without refrigeration, which enhances the applicability of hydrogel systems in resource-limited on-site virus detection. In addition, hydrogels can be easily shaped using injection or printing methods, making them suitable for use in miniaturized diagnostic devices.

The performance of LAMP reactions is strongly influenced by uniform reagent mixing and diffusion control. Hydrogels meet these criteria by providing a structurally compatible medium that can physically entrap primers, enzymes, and deoxynucleotide triphosphates, thereby improving the reaction stability and reproducibility. Furthermore, the local confinement of amplification products within the hydrogel matrices minimizes signal leakage and effectively suppresses nonspecific background noise, which is often observed in solution-phase reactions. These features suggest that hydrogels can function not only as passive reaction matrices, but also as compartmentalized structures suitable for digital LAMP applications.

By exploiting these physical and chemical properties, several hydrogel-based LAMP platforms have been developed in recent years [62-66]. Wang et al. developed an in situ singlecell HPV DNA detection system using a PEG-acrylate hydrogel matrix (Fig. 5 (a)) [62]. This platform was designed to analyze approximately 1,000 cervical cancer cells in parallel. By performing LAMP reactions within the hydrogel, HPV DNA from individual cells can be detected visually. The assay requires only a hotplate for temperature maintenance and a smartphone for readout, thus completing the entire analysis within 30 min. The system demonstrated perfect agreement with quantitative PCR (AUC = 1.00) of 40 clinical samples, confirming its clinical utility.

Fig. 5. 
Representative examples for hydrogel-based LAMP. (a) A large-scale in situ hydrogel-LAMP assay enabling single-cell HPV DNA detection in cervical exfoliated cells using only a hotplate and smartphone. Adapted from Ref. [62]. (b) Nanoconfined LAMP within a polyacrylamide hydrogel matrix enables label-free digital detection by visible Mg₂P₂O₇ colonies, supporting smartphone-based quantification. Adapted from Ref. [65].

Fang et al. developed a nanoconfined digital LAMP platform based on polyacrylamide hydrogels (Fig. 5 (b)) [65]. In this system, the LAMP reaction occurs within ionic polymer hydrogels, allowing for the visual detection of Mg₂P₂O₇, a byproduct of amplification, within individual compartments. The nanoconfined structure of the hydrogel enhanced reaction kinetics and increased light scattering from the Mg₂P₂O₇ particles, enabling the visualization of white colony-like spots without the need for fluorescent labeling. These spots were quantified using smartphone imaging coupled with deep-learning-based image analysis, achieving a counting accuracy of 94.3%. These results demonstrate that high-sensitivity, high-precision, and label-free digital quantification are feasible, underscoring the potential of hydrogel-based systems for on-site molecular diagnostics.

Thus, hydrogels have evolved beyond their role as inert physical scaffolds into multifunctional matrices to enhance amplification efficiency, improve detection specificity, and support digital quantification. However, this approach has several technical limitations. These include reduced reaction speed due to diffusion constraints, challenges in efficiently immobilizing enzymes and primers, and inconsistent reproducibility. Future progress will require the development of novel hydrogel materials, structural optimization, and automated fabrication techniques to establish more robust and practical hydrogel-based LAMP platforms for on-site diagnostic applications.