**4. Discussion**

Advances in diagnostic techniques have allowed clinicians to monitor disease severity in a rapid and noninvasive fashion. With regard to diagnosis of patients with ocular disease, P-ELISA provided a fast and sensitive VEGF assay of aqueous humor to monitor diseases such as senile cataracts, proliferative diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion [17]. P-ELISA has also demonstrated the capacity for complex material collection capacity and multivalue measurement. It has, for instance, been used for outer macromolecule elimination and inner cervicovaginal fluid absorption to detect lactate concentration, glycogen concentration, and pH value in female genital diseases [19]. For patients with bullous pemphigoid, P-ELISA also delivered a simpler and faster diagnostic tool for detection of noncollagenous 16A (NC16A). By examining NC16A concentration with P-ELISA, bullous pemphigoid presence and disease state can be easily identified [20].

Previous concepts for burn depth assessment depend on measurements of tissue perfusion [24]. Laser Doppler imaging (LDI) studies are one of the most popular clinical techniques to assess burn depth. LDI is advantageous due to high accuracy and reduced invasiveness [25–27]. However, most clinical experiences using these modalities do not include examinations with intact blisters, which is a confounding factor that may skew analyzed results.

In this study, we sought to determine the burn depth by measuring angiogenin levels in burn blister fluid using the P-ELISA method. Evaluation of burn fluid contents by conventional ELISA has been successfully used to measure burn depth [9]. However, the necessity for longer operation duration severely hampered plate ELISA techniques for clinical practice. P-ELISA features operation process advantages compared to conventional plate ELISA. This P-ELISA technique required only 15 μL of reagen<sup>t</sup> and an hour of processing compared to the 550 μL and 7 h–8 h required for conventional plate ELISA. Additionally, conventional plate ELISA requires a plate reader and P-ELISA results can be

recorded with a camera (Table 1). Altogether, P-ELISA o ffers clear advantages for rapid and minimally invasive burn depth diagnosis and wound management. It may provide an easy and cost-e ffective method for any healthcare provider to assess burn wounds with excellent diagnostic precision and without the obstacle of a learning curve. However, P-ELISA is still conducted in a qualitative manner till now. Further refinement is needed to improve our device.


**Table 1.** Comparison of paper-based ELISA and conventional plate ELISA.

Wound pH may a ffect the healing process. A lower pH value was reported to be favorable for wound healing [28]. One study showed that healing burn wounds displayed a lower pH level (7.32) compared to unhealing burn wounds (pH 7.73) [29]. We observed an elevated alkaline pH value (8–9) in our burn fluid samples (Table S1). Aspiration of burn fluids in the early stages of injury may be responsible for this phenomenon. This finding is also consistent with a previous study showing that initial stages of healing generated a more basic pH compared to the relatively acidic environment of a repaired wound [30].

We feel that P-ELISA shows grea<sup>t</sup> promise for potential use in clinical practice. Although the clinical application of P-ELISA in burn wound assessment remains to be further studied, we look forward to seeing the application of this new technique in burn wounds. More cases are needed to verify our hypothesis prior to clinical adoption. Further optimization of the paper pattern and additional clinical trials will improve and advance the process and its implementation as a possible tool for healthcare.
