Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens

Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens Xiangwei Zhao, Yu Lu, Chang Liu, Qifan Zhou, Yan Huang, Jingwei Yi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6336864/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The diagnosis of respiratory viral infection via reverse transcription-polymerase chain reaction (RT-PCR) is typically conducted in centralized laboratories using bulky equipment costing 1–2 h. To prevent the spread of infectious diseases, there is an urgent need for ultrafast and accessible molecular diagnostic tools for point-of-care testing (POCT). Here, we developed an ultrafast, POC molecular diagnostic PCRstrip capable of detecting influenza A RNA with high sensitivity (1 copy/µL) in just 15 min. Our system integrated reverse transcription, rapid thermocycling and visual detection at ultralow cost. Broadband absorption plasmonic fabric (BAPF) with excellent photothermal effect was fabricated for volumetric photothermal heating excited by a cost-effective white LED. After photothermal PCR, amplification products can be visually detected by a lateral flow paper strip. Clinical validation of 80 nasopharyngeal swab samples collected from patients suspected of influenza A infection demonstrated a clinical sensitivity of 98.4% and specificity of 94.7%. This fast, ultralow-cost and reliable molecular diagnosis strategy is conducive to POCT, offering an effective tool for non-trained personnel to detect and control infectious diseases individually. Biological sciences/Biotechnology/Assay systems Biological sciences/Biological techniques/Sensors and probes/Surface plasmon resonance Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures Physical sciences/Nanoscience and technology/Nanoscale devices/Nanophotonics and plasmonics Physical sciences/Engineering/Biomedical engineering plasmonic photothermal photonic PCR broadband absorption POCT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Rapid and reliable POCT devices are critical during pandemics, as they enable timely disease containment and reduce fatality rates through decentralized diagnostics accessible to untrained personnel 1 , 2 . While lateral flow assays (LFAs) have emerged as a recognized POCT technology due to their low cost, rapid detection, simple operation and adaptability for home-use, their limited sensitivity severely compromises early-stage pathogen detection when viral loads are lowest yet most critical for transmission control. Currently, PCR has been the gold standard for viral confirmation due to its high sensitivity and specificity 3 – 6 . However, most PCR tests rely on centralized laboratories, sophisticated thermocycling equipment and skilled operators, which hinder its adoption as an efficient on-site screening tool 7 , 8 . To address these limitations, various nucleic acid amplification technologies such as loop-mediated isothermal amplification (LAMP) 9 , 10 , recombinase polymerase amplification (RPA) 11 , 12 and rolling circle amplification (RCA) 13 , 14 have been developed to eliminate the need of bulk and expensive thermocycler and shorten assay time for POCT 15 – 17 . Nevertheless, some of these methods are less established than conventional PCR arising from requiring complex primer design, non-specific amplification and may have inferior accuracy 18 – 20 . Photonic PCR, leveraging plasmonic photothermal effect for ultrafast thermocycling, has been spotlighted as a next-generation PCR system, overcoming key limitations of conventional PCR such as reliance on sophisticated instruments, high energy consumption and prolonged amplification time 18 , 21 , 22 . Recently, researchers have developed several photonic PCR systems based on Au film 23 – 25 , colloidal nanoparticles (NPs) 18 , 26 , 27 , two-dimensional (2D) materials 28 , 29 and carbon black 30 . Although these systems significantly shorten thermocycling time and advance PCR toward POCT, some limitations remain. On the one hand, high-cost lasers were required in some systems to match narrow plasmonic resonance band. On the other hand, large temperature difference may appear in planar heating-type plasmonic thermal cycler with large reaction volume, potentially compromising denaturation efficiency and promoting undesired primer-dimer formation 21 . Moreover, several methods have been adopted for nucleic acid detection in these systems such as gel electrophoresis 31 , 32 , fluorescent 25 , 27 , 33 – 37 and UV-Vis spectroscopy 38 . Fluorescence detection, while highly sensitive, is prone to interference from bubbles and chamber fog 39 , requires costly equipment 40 , and is susceptible to photobleaching. Similarly, gel electrophoresis and UV-Vis spectroscopy require additional post processing or expensive equipment which would length the total assay time and conceal the advantage of rapid thermocycling. Therefore, a volumetric heating-type broadband absorption material could be considered to reduce the cost of light source and improve temperature uniformity. Additionally, lateral flow paper strip could also be considered to detect amplified products in photonic PCR for POCT, which would significantly shorten the total assay time, strengthen the advantages of rapid thermocycling and simplify the workflow. Here, we developed a rapid plasmonic photothermal RT-PCR system for influenza A diagnosis, integrating plasmonic thermocycling and visual detection on single PCRstrip. BAPF was synthesized via in situ growth of Au NPs on porous fabric for efficient plasmonic heating. BAPF served as both a PCR solution container and a photothermal converter. Arising from densely packed, variably sized and randomly distributed plasmonic NPs, BAPF enabling strong broadband absorption in the visible-NIR range and rapid photothermal heating. Therefore, a simple and low-cost white light emitting diode (LED) on/off system was sufficient for the rapid thermal cycling for RT-PCR, while amplified products were visually detected on an immune-strip. The total assay of the PCRstrip was completed within 15 min with a limit of detection (LOD) of 1 copy/µL. The proposed photothermal RT-PCR device was further validated by testing nasopharyngeal swab samples collected from 61 influenza A and 19 non-influenza A patients, demonstrating a sensitivity of 98.4% and specificity of 94.7%. This fast, simple, accurate and cost-effective device may be a promising candidate for POCT and even for general population to use at home. 2. Experiment 2.1 Materials and methods Tetrachloroaurate (III) trihydrate (HAuCl 4 ), trisodium citrate (TSC), thionine, (3-Aminopropyl) trimethoxysilane (APTMS), sodium silicate, bovine serum albumin (BSA), Triton X-100 and sucrose were purchased from Sigma (St Louis, MO, USA). Tri(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, 1 M, pH 8.5), DNA ladder, agarose, λ-DNA, 2 M TAE buffer, streptavidin (SA) and BSA-biotin were provided by Sangon Biotech Co., Ltd (Shanghai, China). 6-FAM monoclonal antibody (10 mg/ml) was obtained from OTwo Biotech (Guangzhou, China). RNA/RNAse free water was purchased from Tiangen (Beijing, China). DL1,000 DNA Marker, TaKaRa Z-Taq™ and One Step PrimeScript RT-qPCR Mix (2X) were received from Takara Biotech. Primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China) (Table S2). Influenza A RNA solution was gained from HZBio (Wuhan, China). STM32F407 microcontroller, white LED and LED driver were purchased from Alientek electronic Co., Ltd (Shenzhen, China), Philips and Telesky electronic Co., Ltd (Shenzhen, China), respectively. MAX31855, cooling fan, TFT LCD touch screen, Lithium battery and relay were gained from Risym electronic Co., Ltd. (Shenzhen, China). Glass fiber (GF-08), sample pad, conjugate pad, absorption pad and backing pad were obtained from Jie Ning Bio Co. Ltd. (Shanghai, China). The nitrocellulose (NC) membrane was received from Whatman-GE Healthcare Company (UK). PET high-temperature tape and Kapton tape were bought from Taobao. Type-K thermocouple was obtained from KAIPUSEN (Jiangsu, China). The thermal images of BAPF were measured by an infrared thermographic camera (ETS320, FLIR). The morphology of BAPF was characterized by scanning electron microscopy (SEM; Zeiss, Ultra Plus). The absorption spectra of BAPF were measured with a UV-1900i UV-Vis spectrophotometer (Shimadzu, Japan). Preparation of BAPF GF-08 was cut into 25 mm × 25 mm and washed twice with deionized water. After drying, glass fiber (GF) was immersed into a mixture of 1 ml of 1% (w/v) HAuCl 4 and 1 ml of 100 mg/ml sodium citrate for 1 h. Washed away excess Au NPs, the GF color turned from white to black, which indicated the successful preparation of BAPF. Then, BAPF was immersed in 2% (v/v) APTMS in ethanol overnight for silanization. Washed by ethanol and water thoroughly, BAPF was treated with aqueous sodium silicate (0.054 wt%, pH 10 adjusted by 0.1 M NaOH) at 95°C for 1 h. Finally, BAPF was taken out, washed and dried. Finally, BAPF was cut into 5 mm × 5 mm and stored for further use. Preparation of PCR reaction mixture The PCR reaction mixture of λ-DNA (total volume of 10 µL) consisted of 0.75 U TaKaRa Z-Taq, 0.3 µM of forward primer, 0.3 µM of reverse primer, 1 µL 10× Z-Taq Buffer (Mg 2+ Plus, 30 mM), 0.8 µL dNTPs, 0.5 µL 10 mg/ml BSA, 0.5 µL λ-DNA and certain amount of RNase-free water. RNA/RNAse free water was used as the template in negative control. The RT-PCR reaction mixture of influenza A RNA (total volume of 10 µL) consisted of 5 µL of PrimeScript 1 Step Enzyme Mix, 0.4 µM of forward primer, 0.4 µM of reverse primer, 1 µL of influenza A RNA and certain amount of RNase-free water. RNA/RNAse free water was used as the template in negative control. Inhibitory effect of fabrics Fabrics were cut into 5 mm × 5 mm squares and individually placed into PCR tubes containing 10 µL of PCR reaction mixture with λ-DNA templates. Each fabric was completely immersed in the solution and subjected to amplification in a conventional benchtop thermocycler. Subsequently, the amplified PCR products were analyzed by gel electrophoresis. PCR amplification protocols in Benchtop instruments Conventional PCR was carried out in an Applied Biosystems SimpliAmp thermal cycler (Thermo Fisher Scientific) following the protocols: 40 cycles of 98°C for 1 s and 60°C for 2 s. Conventional RT-PCR was conducted with following cycling conditions: 52°C for 5 min, 95°C for 10 s and 40 cycles of 95°C for 5 s and 60°C for 30 s. Quantitative reverse transcription PCR (RT-qPCR) was performed using the Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific). For reference RT-qPCR, samples with cycle threshold (C t ) values above 35 were classified as negative. RNA extraction from clinical samples Clinical nasopharyngeal swab samples from influenza A patients were collected by Nanjing First Hospital and preserved in viral transport medium. Viral nucleic acids were extracted from 2 mL of viral preservation solution using an automatic nucleic acid extraction system (SSNP-2000B, Jiangsu Bioperfectus Technologies Co., Ltd.) with corresponding viral nucleic acid extraction kit (SDK60104). The extraction procedure, conducted in a Biosafety Level 2 (BSL-2) laboratory, consisted of 6 sequential steps: lysis, mixing, washing 1,2, elution and release, with a total processing time of approximately 45 min. The extracted RNA samples were subsequently analyzed by RT-qPCR to determine C t values (Table S4) using following thermal cycling protocol: reverse transcription at 52°C for 5 min, initial denaturation at 95°C for 10 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. All RNA samples were stored at -80°C for subsequent analysis. This study received ethical approval from the Ethics Review Committee at Nanjing First Hospital (KY20230807-01-KS-01). Gel electrophoresis analysis PCR products were electrophoretically separated on a 2% (w/v) agarose gel containing 1× GelRed. Electrophoresis was performed at a constant voltage of 125 V for 35 min in 0.5× TAE (Tris/Acetic/EDTA) running buffer. Gel images were recorded by a GenoSens2150 Imaging system (CLINX Science Instruments, China). Preparation of the nucleic acid detection strip The nucleic acid detection strip was composed of sample pad, conjugate pad, NC membrane, absorbent pad and backing pad. Sample pad, conjugate pad, NC membrane and absorbent pad were fixed onto the backing pad in order. During the assembly process, the ends of each part overlapped each other with about 2 mm to ensure continuous fluid flow by capillary effect. 6-FAM monoclonal antibody (1 mg/ml) and BSA-biotin (1 mg/ml) were fixed on the T line and C line, respectively. Finally, the assembled card was dried at 37°C for 1 h and subsequently cut into 3-mm wide test strips using a programmable cutting machine. The prepared strips were then stored in a desiccator for future use. 1 ml of 35 nm Au NPs was centrifuged at 7000 rpm for 10 min and resuspended in 0.5 ml of deionized water. Then, 8 µL of SA solution (1 mg/ml) was added and shaken gently at room temperature for 30 min. Next, 50 µL of BSA solution (10% w/v) was added and incubated for 2 h to block nonspecific binding sites. Then, the conjugated Au NPs was centrifuged 7000 rpm for 10 min and resolved in 50 µL of resuspending solution (1% BSA, 15% sucrose and 0.2% Triton X-100 in 20 mM Tris-HCl). Finally, Au NP/SA nanotag was impregnated onto the conjugated pad of paper strip and dried. 3. Results and discussion Principle of the PCRstrip The working principle of the PCRstrip is illustrated in Fig. 1 , where nucleic acid amplification and detection are integrated into a handheld device. The BAPF containing the PCR reaction mixture was mounted at the front of the PCRstrip to enable ultrafast photothermal cycling. When illuminated by a white LED, photons excited plasmonic electrons on BAPF, generating hot electrons within 100 fs. These hot electrons, with low heat capacity, rapidly raised the temperature to several thousand degrees Kelvin, heating the surrounding PCR solution effectively. A microcontroller was employed to remotely control the LED via pulse-width modulation (PWM), precisely regulating irradiation intensity, duration and cooling cycles. Compared with laser-based systems, white LED consumed significantly less power and reduced the device cost, making it ideal for POCT. After photothermal amplification, PCR products were released and flowed onto the paper strip for visual detection. The liquid migrated through the sample pad, conjugated pad, T line and C line. Target nucleic acids, amplified with end-labeled primers, bound to SA-functionalized Au nanotags (SA-Au nanotags), forming SA-Au nanotags-DNA complexes. At the T line, anti-FAM antibodies captured 6-FAM-labeled primers, producing a visible SA-Au nanotag-DNA-anti-6-FAM complex. In the absence of amplification products, Au nanotags were captured only on the C line. Therefore, the presence of target nucleic acid can be analyzed by observing the T line color. The entire process, including RT-PCR and visual detection, was completed within 15 min, facilitating rapid and accessible large-scale screening for POCT during pandemics. Characterization of BAPF The BAPF functioned as both a stable PCR solution container with high surface area and an efficient photothermal material. BAPF was prepared by in situ synthesis of Au NPs on GF (Fig. 2 a). In this process, HAuCl 4 and TSC would infiltrate the interspace of GF via capillary effect, reducing Au 3+ to Au NPs on GF over time. The BAPF exhibited strong broadband light absorption in the visible range, enabling rapid photothermal conversion and efficient heat transfer to the surrounding PCR solution. Meanwhile, GF exhibited a three-dimensional multi-interspace structure with pores at micrometer scale, which provided favorable condition for efficient accommodation and uniform distribution of the PCR reaction solution (Figure S1 ). Moreover, the highly porous structure facilitated internal mass transfer required for an effective reaction. Therefore, BAPF functioned as volumetric heating source rather than planar heating source. Since the solution was contained in the membrane, the surface area for heating per solution volume was higher than that in the PCR tube 41 . Plasmonic heating could effectively heat the whole PCR solution and the heating rate was increased greatly. In fact, the photothermal performance of BAPF strongly depended on the loading amount of Au NPs. Therefore, the reaction time (10, 20, 30, 45, 60 and 120 min) for BAPF was firstly optimized. As the reaction time increased, the loading amount of Au NPs increased, turning the BAPF from light gray to black. SEM images in Fig. 2 b confirmed that the loading amount of Au NPs on GF increased initially and saturated over 60 min. The optical absorbance spectra of six BAPFs were shown in Fig. 2 c. All BAPFs exhibited broad and flat absorbance in the visible light range may arising from the hybridization localized surface plasmon (LSP) modes from nanometer-sized Au nanoclusters with randomized sizes and positions, resulting in disordered plasmonic broadband absorption performance 42 – 44 . The absorbance of BAPF increased with reaction time as more Au NPs formed and densely packed on the fabric, however, saturated over 60 min. Since the BAPF was a strong absorber of light over full spectrum of visible light, a cost-effective white LED was used for photothermal conversion. Upon illumination, the temperature of BAPF increased rapidly, heating the entire sample uniformly (Fig. 2 d, 2 e). In contrast, the pristine GF barely sensed the white light, resulting in unnoticeable temperature changes under identical condition. The marked contrast further confirmed the thermoplasmonic mechanism underlying the observed thermal response in BAPF. To assess the photothermal effect dependence on the Au NPs loading amount, six BAPF samples synthesized with different reaction time were illuminated by the white LED for a certain time and cooled (Fig. 2 f). The heating rate increased initially and saturated over 60 min which was in coincidence with optical absorbance. Therefore, the reaction time of BAPF was optimized at 60 min for subsequent experiments. To further evaluate the photothermal responsiveness of BAPF, temperature profiles of BAPF illuminated by LED modulated by different PWM duty cycles were shown in Fig. 2 g. These results demonstrated precise control over heating dynamics achievable with the BAPF, making it an ideal candidate for ultrafast photothermal PCR applications. Inhibitory effect was also a significant factor to be considered. The inhibitory effect of fabrics on PCR was investigated and shown in Fig. 2 h. GF shown barely no adverse effect on PCR which made it a good candidate for containing PCR solution. However, BAPF shown great inhibitory effect on PCR arising from the strong adsorption of polymerase on Au NPs 45 , 46 . Therefore, a hermetic silica shell was coated on Au NPs to eliminate PCR inhibition arising from polymerase adsorption. To ensure that the silica shell is pinhole-free, SERS spectra of thionine were measured on BAPF and silica coated BAPF, respectively. As shown in Fig. 2 i, the overall shape of 1350 to 1450 cm − 1 region in the spectra of thionine on silica coated BAPF was similar with that in the ordinary Raman spectra. The intensity of 1120 cm − 1 was relatively low in the ordinary Raman spectra and the spectra on silica coated BAPF, whereas high in the SERS spectra on BAPF without silica shell. Above differences were in consisted with that in reported results, proving the shell was pinhole-free 47 . Obviously in Fig. 2 h, BAPF passivated with silica shown negligible inhibitory effect on PCR owing to the imporous silica coating. In addition, the silica shell offered Au NPs with thermal stability against the reshaping of the nanoparticle (Figure S2). Moreover, the optical image of large-scale-manufactured-BAPF (110 mm × 110 mm) was shown in Figure S3, which can be divided into 484 individual 5 mm × 5 mm units. Consistent photothermal performance was observed in six batches of BAPF (Figure S4), proving the excellent reproducibility and process stability. Therefore, BAPF as an excellent plasmonic photothermal material can be reliably produced in a high-throughput scale. Numerical simulation The temperature difference is a critical factor affecting the accuracy and reproducibility of plasmonic photothermal nucleic acid amplification 21 . As a proof-of-concept, a numerical simulation employing finite element method (FEM) was performed in COMSOL Multiphysics 5.6 to compare the temperature distribution of photothermal heating of solution in planar plasmonic Au substrate and BAPF. The model parameters of planar plasmonic Au substrate were adopted from a reported paper 32 , consisting of a 120 nm-thick Au membrane. The detailed model structure was shown in Figure S5 and Table S1 . For the model of BAPF, the structure was simplified as orthogonal stacked layers of Au-coated SiO 2 fiber, with each fiber having a diameter of 20 µm and a center-to-center distance of 50 µm between adjacent layer. The detailed structural design and geometric parameters were shown in Figure S6 and Table S1 . Two physics Electromagnetic Waves and Heat Transfer in Solids and Fluids were coupled together to calculate the resulting temperature illuminated by a light beam. Numerical simulation results were shown in Fig. 3 a and 3 b. When the Au film was used as the heating element, heat was transferred from the bottom upward, leading to reduced heating uniformity. In contrast, BAPF exhibited a three-dimensional multi-interspace structure with micrometer-scale pores that allowed volumetric heating, resulting in even heat distribution throughout the reaction mixture. As obviously illustrated in Fig. 3 c, the temperature difference in BAPF exhibited the maximum difference 0.6°C, while that was 2.5°C in Au film. Therefore, the homogeneity of heating is greatly improved in BAPF, which is conducive to preventing the decline of denaturation efficiency and the undesired primer-dimer formation. Moreover, the use of BAPF as a solution container increased the surface area-to-volume ratio for heating and cooling 41 . The numerical simulation showed that the solution in BAPF took 1.7 s to reach 98°C from 20°C, whereas that in the Au membrane took 2.5 s (Fig. 3 d). Similarly cooling of solution in BAPF took 170 s to reach 20°C from 98°C, whereas that in the Au membrane took 330 s (Fig. 3 e). These results demonstrated that BAPF enabled volumetric heating, facilitating significantly faster thermocycling while ensuring uniform thermal distribution across the entire sample volume with minimal thermal gradients. Photothermal PCR in BAPF The photothermal PCR performance of BAPF under white LED illumination was firstly investigated using λ-DNA as the template. A piece of BAPF containing 10 µL of PCR solution was sealed by two pieces of transparent PET tape to prevent evaporation during thermocycling (Fig. 4 a). Noticeably, the PET tape was incubated by 10% (w/v) BSA in advance to prevent nonspecific adsorption of polymerase. A thin thermocouple was attached tightly onto BAPF to measure the temperature for closed-loop control. Meanwhile, signals related to real-time temperature were transmitted to a microcontroller through a MAX31855 thermocouple module. Then, the sealed BAPF was mounted right above a white LED for thermocycling. STM32F407 with embedded code controlled the LED irradiation intensity and fan on/off through I/O port, enabling precisely controlled thermocycling. Stable thermocycling results of 40 cycle PCR within 373 s were shown in Fig. 4 b, with the heating rate of 11.875°C/s and cooling rate of 8.137°C/s. A typical temperature profile of a single thermal cycle was shown in Fig. 4 b, the heating stage from 60°C to 98°C was realized by white LED illumination and the cooling stage was accomplished by white LED off and fan on. 100 pieces of BAPF were randomly selected to perform 40 cycles with annealing/extension at 60°C and denaturation at 98°C (Fig. 4 c). The temperature fluctuations were minimal, with standard deviations of 1.35°C at 98°C and 0.3°C at 60°C, proving the high consistency and reliability of photothermal temperature control achieved by BAPF. Henceforth, the BAPF can be used for subsequent nucleic acid amplification applications. After photothermal PCR, the amplification products were extracted by centrifugation for gel electrophoresis analysis. The electrophoretic band of λ-DNA (98 bp, Table S3) amplified by benchtop device and BAPF were shown in Fig. 4 d, demonstrating matching band and comparable amplification performance. The fluorescence intensity of each electrophoretic band was quantified using ImageJ software. Compare to the PCR products obtained from a conventional PCR thermocycler, the photothermal amplification yield exceeded 85%. In addition, the photothermal PCR exhibited reproducible DNA amplification results, as shown in Fig. 4 e. PCR products from eight independent photothermal PCR tests shown similar gel electrophoretic band intensity. Gel electrophoresis analysis results of λ-DNA dilutions (0-0.1 ng/µL) after photothermal PCR were shown in Fig. 4 f. The band intensity gradually decreased as the λ-DNA concentration reduced from 0.1 ng/µL to 0, with a visual detection limit of approximately 0.1 pg/µL. These results demonstrated that DNA can be efficiently amplified in BAPF by photothermal PCR, confirming the feasibility of proposed strategy. Fabrication and working principle of photothermal PCR chip The photothermal PCR chip (107 mm × 44 mm × 11 mm) was developed by integrating the BAPF and lateral flow paper strip in a customized three-dimensional (3D) printing plastic housing for on-site rapid diagnosis (Fig. 5 a). The casing had an opening in the front and the other in the middle. The front opening was designed to expose BAPF for light illumination and fan cooling. The other opening was for visual observation of nucleic acid paper strip. BAPF was sealed by two pieces of PET tape and mounted in the front of the chamber. Subsequently, a lateral flow paper strip was fixed in the middle rear of the chamber. Importantly, a 2 mm overlap was kept between BAPF and conjugated pad to ensure amplified products released from BAPF flowed onto test strip through capillary force successfully. RT-PCR is currently the most sensitive and specific detection method of viral RNA 48 . To assess whether this photothermal amplification strategy is also applicable to RNA targets, RT-PCR has also been carried out with influenza A RNA. The on/off duty of white LED was slightly modified to conduct thermocycling for RT-PCR. The RT-PCR protocol began with a constant temperature at 52°C for 3 min (< 1°C variation) for sufficient complementary DNA (cDNA). Then, 40 cycles of PCR amplification were conducted between 95°C and 60°C for denaturation and annealing/extension (Fig. 5 b). The entire RT-PCR can be finished within 10 min in proposed photothermal PCR chip. M1 gene (123 bp) of influenza A RNA was used as a positive control to demonstrate the capability of proposed photothermal RT-PCR chip (Table S3). The photothermal RT-PCR products of various concentrations of Influenza A RNA were analyzed by gel electrophoresis, with fluorescence intensity correlating with RNA concentration (Fig. 5 d). For visual detection of Influenza A RNA, the PET tape was punctured after photothermal RT-PCR to release amplified products onto the lateral flow paper strip (Figure S7). Consequently, PCR products labeled with biotin and 6-FAM at opposite ends would form complexes with SA-Au nanotags (Table S2). Complexes were immobilized on the T line due to the affinity of 6-FAM and anti-6-FAM antibody. Figure 5 e shown the lateral flow paper strip detection results of different concentrations of influenza A RNA after ultrafast photothermal RT-PCR. The color intensity of T line varied with the RNA concentration, achieving a visual detection limit of approximately 1 copy/µL. Additionally, test strips for six repetitions of influenza A RNA photothermal RT-PCR products demonstrated excellent reproducibility and accuracy (Figure S8). Therefore, this BAPF-based photothermal chip was also applicable for RNA targets with high sensitivity and stability. Clinical diagnosis of nasopharyngeal swab samples on POCT device To enable POCT applications, an integrated opto-mechatronic system was developed to enhance the portability and functionally of the device (Fig. 6 a). The POCT device measured 107 mm × 96 mm × 103 mm and weighed 437 g, with all optoelectronic components encapsulated in a custom 3D-printed housing for automatic PCR operation. The system consisted of several key components that work in concert for precise and efficient thermocycling. A white LED, serving as the photothermal excitation source, was mounted on a cooper radiator equipped with a cooling fan to dissipate heat generated during operation. Above the BAPF, another 12 V fan was installed to accelerated the cooling rate during thermocycling process, enabling rapid temperature transitions between heating and cooling phases. The microcontroller unit (MCU), LED driver and relay were integrated on a printed circuit board (PCB). As illustrated in the control schematic, the MCU implemented a digital proportional-integral-derivative (PID) temperature control algorithm to precisely regulate the thermocycling process (Fig. 6 b). The MCU monitored temperature via a type-K thermocouple, continuously calculated the error between the preset and measured values, and dynamically adjusted output variables through PWM to maintain the desired temperature profile. This closed-loop control mechanism ensured high accuracy and reproducibility in temperature regulation. For user interaction, a TFT LCD touch panel provided an intuitive graphical interface, allowing users to input PCR parameters (e.g., cycle number, temperature settings) and display real-time temperature curves during operation (Figure S9). For remote operation, a mobile application was developed to communicate wirelessly with the instrument via Bluetooth. The application, installed on a smartphone, featured a user-friendly interface for inputting amplification parameters and real-time display of amplification progress (Figure S10). The whole device was powered by a rechargeable lithium battery, making it highly portable and suitable for POCT applications. To initiate operation, the user simply turned on the power button, inserted the photothermal chip preloaded with PCR solution, and input PCR parameters via the touch panel or mobile app. Once started, the system automatically executed ultrafast photothermal thermocycling, with the MCU coordinating the LED, fans and sensors to ensure optimal performance. To validate the clinical applicability of our system, we conducted additional tests using the palm-size plasmonic photothermal PCR instrument. Nasopharyngeal swab samples collected from patients infected with influenza A virus were analyzed using the customized prototype device. A total of 80 human saliva specimens (61 positives and 19 negatives) were tested. The C t values of all samples were confirmed in advance using a conventional benchtop RT-qPCR after the extraction of viral RNA from the swabs (Table S4 and Fig. 6 c). The whole workflow, illustrated in Fig. 6 d, included sample collection, RNA extraction, sample loading, photothermal PCR and detection. The photothermal RT-PCR protocol included an initial reverse transcription at 52°C for 3 min, followed by 40 amplification cycles of denaturation at 95°C and annealing/extension at 60°C. Upon completion of the RT-PCR final cycle, amplicons were released and the results could be visually assessed using lateral flow strips by naked eyes. The resulting images of lateral flow paper strips were shown in Figure S11 and the T line intensity of all samples were mapped in Fig. 6 e. Using proposed photothermal RT-PCR device, 60 of 61 positive samples were identified, while all the negative controls except one were identified correctly. Noticeably, positive samples exhibited significantly higher levels of T line intensity than negative samples (Fig. 6 f). The clinical performance of proposed photothermal RT-PCR showed a diagnostic agreement of 96.5% compared to the conventional RT-qPCR and demonstrated a clinical sensitivity and specificity of 98.4% and 94.7%, respectively. To further compare the performance of the POC device with conventional RT-qPCR, normalized C t values and normalized T line intensity were plotted and linear fitted. The Pearson correlation coefficient (r) was − 0.93, which proved the existence of a correlation between the conventional RT-qPCR and proposed method (Fig. 6 g). Overall, proposed device had the potential to conduct influenza A molecular diagnosis in clinical situation with a significantly short operation time of 15 min. Obviously, nucleic acid testing of other diseases can also be conducted on proposed device after primers design and amplification protocol optimization. This device shown remarkable features, including: (1) the assay was based on well-established RT-PCR rather than isothermal amplification test. (2) the greatly shortened analysis time was conducive to on-site diagnosis. (3) mobile and professional-free operation alleviated the pressure of laboratory testing during pandemic. (4) ultralow cost was beneficial to POC testing (Table S5). (5) naked eye detection greatly saved detection time. According to these prominent advantages, our platform is of great potential for personal timely molecular diagnosis of various diseases in POCT. Conclusion In this work, a simple, cost-effective and robust POC photothermal RT-PCR device has been developed for personal pathogen diagnosis in POCT. The device relied on a small photothermal chip and a portable photothermal instrument, which accomplished rapid amplification and naked-eye detection without additional expensive PCR infrastructure and power supply. Plasmonic photothermal nanostructure and lateral flow paper strip were integrated in single PCRstrip for ultrafast nucleic acid amplification and visual detection. BAPF which exhibited strong and broadband absorption in entire visible range produced ultrafast nanoplasmonic heating resulting from strong light-to-heat conversion of white LED. Programmable and automatical thermocycling was completed in a customized compact prototype machine within 15 min (10 min for RT-PCR and 5 min for detection). The clinical diagnostic performance of proposed device was validated with sensitivity and specificity of 98.4% and 94.7%. Proposed PCRstrip combines advantages of low cost, rapid amplification time and compact size, making it a promising tool for POC molecular diagnosis in resource-limited or personal situations in against of pandemic and for the distinction diagnosis with other respiratory viruses. Declarations Acknowledgements The authors gratefully acknowledge the financial supports from National Natural Science Foundation of China (82361138570, 82402755), Shenzhen Science and Technology Plan Project (JCYJ20230807114610021) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX22_0246). References Jung WE, Han J, Choi J-W, Ahn CH (2015) Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies. Microelectron Eng 132:46–57 Xiao M et al (2022) Virus Detection: From State-of-the-Art Laboratories to Smartphone-Based Point-of-Care Testing. Adv Sci 9 Horejs C (2021) Artificially intelligent nanopore for rapid SARS-CoV-2 testing. Nat Rev Mater 6:650–650 Kevadiya BD et al (2021) Diagnostics for SARS-CoV-2 infections. Nat Mater 20:593–605 Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582 Yolshin ND et al (2022) Detection of the Omicron SARS-CoV-2 Lineage and Its BA.1 Variant with Multiplex RT-qPCR. Int J Mol Sci 23 Marx V (2015) PCR heads into the field. Nat Methods 12:393–397 Petralia S, Conoci S (2017) PCR Technologies for Point of Care Testing: Progress and Perspectives. ACS Sens 2:876–891 Artik Y et al (2022) Comparison of COVID-19 laboratory diagnosis by commercial kits: Effectivity of RT-PCR to the RT-LAMP. J Med Virol 94:1998–2007 Huang X, Tang G, Ismail N, Wang X (2022) Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva. Ebiomedicine 75 Cherkaoui D et al (2022) Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2. Diagnostics 12 Luo Z et al (2022) Digital recombinase polymerase amplification in hydrogel nanofluidic chip for ultrafast and precise quantification of pathogens in fresh food. Sens Actuators B Chem 367 Ali MM et al (2014) Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chem Soc Rev 43:3324–3341 Choi MH, Kumara GSR, Lee J, Seo YJ (2022) Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification. Anal Bioanal Chem 414:5907–5915 Colbert AJJ et al (2022) PD-LAMP smartphone detection of SARS-CoV-2 on chip. Anal Chim Acta 1203 Dong Y et al (2022) Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe. ACS Sens 7:730–739 Liang L-G et al (2023) Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B. J Med Virol 95 Cheong J et al (2020) Fast detection of SARS-CoV-2 RNA via the integration of plasmonic thermocycling and fluorescence detection in a portable device. Nat Biomed Eng 4:1159–1167 de Paz D, Brotons H, P., Munoz-Almagro C (2014) Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics. Expert Rev Mol Diagn 14:827–843 Kang T, Lu J, Yu T, Long Y, Liu G (2022) Advances in nucleic acid amplification techniques (NAATs): COVID-19 point-of-care diagnostics as an example. Biosens Bioelectron 206 Kang B-H et al (2021) Ultrafast and Real-Time Nanoplasmonic On- Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics. ACS Nano 15:10194–10202 You M et al (2020) Ultrafast Photonic PCR Based on Photothermal Nanomaterials. Trends Biotechnol 38:637–649 Kim KH et al (2024) Plasmonic digital PCR for discriminative detection of SARS-CoV-2 variants. Biosens Bioelectron 246 Kim KH et al (2023) Ultra-Fast Photonic Digital Polymerase Chain Reaction based on N-Heterocyclic Carbene Self-Assembled Monolayer. Adv Funct Mater 33 Seo SE et al (2024) Ultrafast Molecular Diagnosis-Based Solid-Phase Photonic PCR for Respiratory Pathogen Variant Discrimination. Adv Funct Mater 34 Blumenfeld NR et al (2022) Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis. Nat Nanotechnol 17:984– Nam KS, Piri A, Choi S, Jung J, Hwang J (2024) Air sampling and simultaneous detection of airborne influenza virus via gold nanorod-based plasmonic PCR. J Hazard Mater 477 Lee S et al (2025) Ultrafast Photonic PCR with All-Solution-Processed Ti3C2Tx-Based Perfect Absorbers. Laser Photonics Rev Parvin R, Zhang L, Zu Y, Ye F (2023) Photothermal Responsive Digital Polymerase Chain Reaction Resolving Exosomal microRNAs Expression in Liver Cancer. Small 19 Shrestha K et al (2023) Mobile Efficient Diagnostics of Infectious Diseases via On-Chip RT-qPCR: MEDIC-PCR. Adv Sci 10 Lee Y et al (2020) Nanoplasmonic On-Chip PCR for Rapid Precision Molecular Diagnostics. ACS Appl Mater Interfaces 12:12533–12540 Son JH et al (2016) Rapid Optical Cavity PCR. Adv Healthc Mater 5:167–174 Cho B et al (2019) Nanophotonic Cell Lysis and Polymerase Chain Reaction with Gravity-Driven Cell Enrichment for Rapid Detection of Pathogens. ACS Nano 13:13866–13874 Jiao Y, Zhang Z, Wang K, Zhang H, Gao J (2023) Rapid detection of Salmonella in food matrices by photonic PCR based on the photothermal effect of Fe3O4. Food Chem X 19 Kim I et al (2024) Ultrafast Metaphotonic PCR Chip with Near-Perfect Absorber. Adv Mater 36 Wu J et al (2021) A rapid and sensitive fluorescence biosensor based on plasmonic PCR. Nanoscale 13:7348–7354 Zhou X et al (2024) Rapid quantitative detection system constructed via photonic PCR based on the photothermal effect of NH2-MWCNTs/TiO2. Vacuum 229 Mohammadyousef P, Paliouras M, Trifiro M, Kirk AG (2020) in Symposium on Label-Free Biomedical Imaging and Sensing (LBIS) held at SPIE BiOS Conference, Vol. 11251San Francisco, CA Lee SH et al (2019) Bubble-free rapid microfluidic PCR. Biosens Bioelectron 126:725–733 Guevara-Pantoja PE et al (2023) Hybrid Opto-Thermocycler for RT-qPCR Detects SARS-CoV-2. Adv Mater Technol 8 Kim K et al (2023) Rapid PCR kit: lateral flow paper strip with Joule heater for SARS-CoV-2 detection. Mater Horiz 10:1697–1704 Chen J-a et al (2023) Broadband and Spectrally Selective Photothermal Conversion through Nanocluster Assembly of Disordered Plasmonic Metasurfaces. Nano Lett 23:7236–7243 Zhang X-Y et al (2018) Silver nanoplate aggregation based multifunctional black metal absorbers for localization, photothermic harnessing enhancement and omnidirectional light antireflection. J Mater Chem C 6:989–999 Zhou L et al (2017) Self-assembled spectrum selective plasmonic absorbers with tunable bandwidth for solar energy conversion. Nano Energy 32:195–200 Shang L, Nienhaus GU (2017) In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy. Acc Chem Res 50:387–395 Vu BV, Litvinov D, Willson RC (2008) Gold nanoparticle effects in polymerase chain reaction: Favoring of smaller products by polymerase adsorption. Anal Chem 80:5462–5467 Li J-F, Anema JR, Wandlowski T, Tian Z-Q (2015) Dielectric shell isolated and graphene shell isolated nanoparticle enhanced Raman spectroscopies and their applications. Chem Soc Rev 44:8399–8409 Qiu G et al (2020) Dual-Functional Plasmonic Photothermal Biosensors for Highly Accurate Severe Acute Respiratory Syndrome Coronavirus 2 Detection. ACS Nano 14:5268–5277 Additional Declarations There is NO Competing Interest. Supplementary Files SIUltralowcostpersonalPCRstripwithvolumetricheatingbasedonbroadbandabsorptionplasmonicfabricforPOCTofpathogens.docx Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6336864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":443932228,"identity":"3934d3ec-897c-4ef8-a5e4-3e889c62b418","order_by":0,"name":"Xiangwei Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYHACNiC2gTB5SNCSRrqWwyRoMbiR/uzBj4rz9vwSCYwP3rYxyJsT0iI5IyHdsOfM7cSZMxKYDee2MRjubCCghV864ZgEb9vtBIMbCWzSvG0MCQYHCGhhk05sk/zbds7e/kYC+2+itPBLJ4MMP8C4QSKBjZkoLZLzn7FJy5xJTpxx5mGz5JxzEoYbCGkxOHP8meSbCjt7/vbkgx/elNnIE7QFCTA2AAkJ4tWPglEwCkbBKMANACtzOtYvHaAlAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0678-5219","institution":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University","correspondingAuthor":true,"prefix":"","firstName":"Xiangwei","middleName":"","lastName":"Zhao","suffix":""},{"id":443932229,"identity":"0aa20a37-e822-4d4c-bdda-777c0d102ece","order_by":1,"name":"Yu Lu","email":"","orcid":"","institution":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lu","suffix":""},{"id":443932230,"identity":"d1e58d8b-ec0a-4b36-b22a-3dc92275d3f8","order_by":2,"name":"Chang Liu","email":"","orcid":"","institution":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Liu","suffix":""},{"id":443932231,"identity":"13a00ea4-e97b-4412-b3b5-9fe333bafa7e","order_by":3,"name":"Qifan Zhou","email":"","orcid":"","institution":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Qifan","middleName":"","lastName":"Zhou","suffix":""},{"id":443932232,"identity":"32baf89f-d626-481f-9ac8-38788055cb4e","order_by":4,"name":"Yan Huang","email":"","orcid":"","institution":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Huang","suffix":""},{"id":443932233,"identity":"ac67a30e-a522-4378-9335-760fa49aa74d","order_by":5,"name":"Jingwei Yi","email":"","orcid":"","institution":"Jiangsu Bioperfectus Technologies Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Jingwei","middleName":"","lastName":"Yi","suffix":""},{"id":443932234,"identity":"fa389d41-24d6-4333-b21e-9ac7eb5c791b","order_by":6,"name":"Wei Jin","email":"","orcid":"","institution":"Jiangsu Bioperfectus Technologies Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Jin","suffix":""},{"id":443932235,"identity":"dbb4217d-4e1c-45a6-b414-0c1ea8dbfafe","order_by":7,"name":"Zhonghua Liu","email":"","orcid":"","institution":"Jiangsu Bioperfectus Technologies Co., Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Zhonghua","middleName":"","lastName":"Liu","suffix":""},{"id":443932236,"identity":"96feb3c6-89d6-4516-b73d-b597aac6e35b","order_by":8,"name":"Bangshun He","email":"","orcid":"","institution":"Department of Laboratory Medicine, Nanjing First Hospital, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bangshun","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-03-30 05:30:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6336864/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6336864/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81954407,"identity":"f4d63e38-e2dd-48a1-8b27-b1ebe37482b0","added_by":"auto","created_at":"2025-05-05 09:43:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64861,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the PCRstrip\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/a1635e21965702d1d8955841.jpg"},{"id":81954413,"identity":"caedefe5-79c0-4baa-aa9e-e111dac779dc","added_by":"auto","created_at":"2025-05-05 09:43:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145888,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of the preparation of BAPF by in-situ reduction of Au NPs on GF. (b) Optical images and SEM images of BAPFs with different reaction time. The scale bar represents 2 μm. (c) Absorbance spectra of six BAPFs with different reaction time. (d) Thermal images of GF and BAPF illuminated by white LED. (e) Temperature profiles of GF and BAPF illuminated by white LED. (f) Temperature profiles of six BAPFs with different reaction time under white LED. The LED was on at 12 s and turned off after 100 s. (g) Temperature profiles of BAPF under white LED driven by different PWM duty cycles. (h) Screening of GF, BAPF and silica coated BAPF for inhibitory effect. (1: DNA marker; 2: Control; 3: GF; 4: BAPF; 5: silica coated BAPF) (i) Raman spectra of thionine and SERS spectra of thionine onBAPF and silica coated BAPF.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/75dc0f1c79b8952f3138857e.jpg"},{"id":81954409,"identity":"b4520e64-8148-4fc3-9a92-74eedc933ff6","added_by":"auto","created_at":"2025-05-05 09:43:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74714,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature gradient of (a) sample solution on Au membrane and (b) sample solution in BAPF calculated by FEM. (c) Calculated data for the maximum difference of solution temperature on the Au membrane and in the BAPF. (d) Heating and (e) cooling of solution on the Au membrane and in the BAPF.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/912e8e1dca3d1b1ae2e00a82.jpg"},{"id":81954411,"identity":"391a943e-2f7c-405b-b179-686e04bd8f5b","added_by":"auto","created_at":"2025-05-05 09:43:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":131190,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of the working flow of PCR in the BAPF and products detection by gel electrophoresis. (b) 40 thermal cycles of BAPF (range of 60-98 °C) (left); schematic illustration of the repeated thermocycling: 60-98 °C: LED on to generate heat via photothermal effect, 98-60 °C: LED off and fan on for cooling (right) (c) Temperature stability in denaturation (red) and annealing/extension (blue) steps. (d) Gel electrophoresis images of PCR products from a conventional thermocycler and photothermal PCR (left) (Lane 1: Conventional PCR; Lane 2: Photothermal PCR); Fluorescence intensity of PCR products from a conventional thermocycler and photothermal PCR (right). (e) Gel electrophoresis results for reproducibility of photothermal PCR using the λ-DNA template. The upper bar chart represents the fluorescence intensity of each gel lane. (f) Gel electrophoresis detection of different concentrations of λ-DNA after photothermal amplification on BAPF (Lane 1: 0.1 ng/μL; Lane 2: 10 pg/μL; Lane 3: 1 pg/μL; Lane 4: 0.1 pg/μL; Lane 5: 10 fg/μL; Lane 6: 1 fg/μL; Lane 7: Negative control).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/4594b3e4479a089c678be2e0.jpg"},{"id":81955424,"identity":"48f9c45f-efe6-4960-9ff4-bf4cfabc2bbc","added_by":"auto","created_at":"2025-05-05 09:51:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78559,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of the photothermal PCR chip. (b) Digital image of inside and outside of the photothermal PCR chip. (c) Temperature curve for photothermal RT-PCR. (d) Gel electrophoresis and (e) Lateral flow paper strip detection of different concentrations of influenza A after photothermal RT-PCR (Lane 0: DNA marker; Lane 1: 10\u003csup\u003e7\u003c/sup\u003e copy/ml; Lane 2: 10\u003csup\u003e6\u003c/sup\u003e copy/ml; Lane 3: 10\u003csup\u003e5\u003c/sup\u003e copy/ml; Lane 4: 10\u003csup\u003e4\u003c/sup\u003e copy/ml; Lane 5: 10\u003csup\u003e3\u003c/sup\u003e copy/ml; Lane 6: 10\u003csup\u003e2\u003c/sup\u003e copy/ml; Lane 7: 10\u003csup\u003e1\u003c/sup\u003e copy/ml; Lane 8: Negative control).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/0072b97f368b749a9790d887.jpg"},{"id":81955430,"identity":"80941803-361e-405a-b0b8-6c36f5982257","added_by":"auto","created_at":"2025-05-05 09:51:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":135560,"visible":true,"origin":"","legend":"\u003cp\u003ea) Photograph of proposed POC device. (b) Block diagram illustrating the internal components of the instrument. Arrows show the communication direction between the thermocouple, LED, electronics and the MCU. (c) Heatmap showing the results of influenza A RNA detection in 80 nasopharyngeal swab samples using RT-qPCR method. Values represented in the heatmap are the C\u003csub\u003et\u003c/sub\u003e value of each sample detection results. The “+” represents the positive sample confirmed by RT-qPCR, the “−” represents the negative sample confirmed by RT-qPCR. (d) Work flow of influenza A RNA detection based on POC device. (e) Heatmap showing the results of influenza A RNA detection in 80 nasopharyngeal swab samples using POC device. Values represented in the heat map are the T line intensity of each sample detection results. (f) Scatter plot of the test line intensity of 80 samples detected by photothermal RT-PCR method (t test; ****, p \u0026lt; 0.0001). (g) Correlation between RT-qPCR method and proposed POC device.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/521bd610dfd8edea4cbfd014.jpg"},{"id":81956737,"identity":"be7f8045-18d2-4748-bf7e-6f9c51d842b8","added_by":"auto","created_at":"2025-05-05 09:59:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1283852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/f448232d-5de1-46f9-82b5-371150679fed.pdf"},{"id":81954420,"identity":"f89591db-2b4d-47db-b1d3-f4840c0a1768","added_by":"auto","created_at":"2025-05-05 09:43:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10495453,"visible":true,"origin":"","legend":"Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens","description":"","filename":"SIUltralowcostpersonalPCRstripwithvolumetricheatingbasedonbroadbandabsorptionplasmonicfabricforPOCTofpathogens.docx","url":"https://assets-eu.researchsquare.com/files/rs-6336864/v1/5dac6c61232f21f78728b101.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRapid and reliable POCT devices are critical during pandemics, as they enable timely disease containment and reduce fatality rates through decentralized diagnostics accessible to untrained personnel\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. While lateral flow assays (LFAs) have emerged as a recognized POCT technology due to their low cost, rapid detection, simple operation and adaptability for home-use, their limited sensitivity severely compromises early-stage pathogen detection when viral loads are lowest yet most critical for transmission control. Currently, PCR has been the gold standard for viral confirmation due to its high sensitivity and specificity\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, most PCR tests rely on centralized laboratories, sophisticated thermocycling equipment and skilled operators, which hinder its adoption as an efficient on-site screening tool\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. To address these limitations, various nucleic acid amplification technologies such as loop-mediated isothermal amplification (LAMP)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, recombinase polymerase amplification (RPA)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and rolling circle amplification (RCA)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e have been developed to eliminate the need of bulk and expensive thermocycler and shorten assay time for POCT\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Nevertheless, some of these methods are less established than conventional PCR arising from requiring complex primer design, non-specific amplification and may have inferior accuracy\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePhotonic PCR, leveraging plasmonic photothermal effect for ultrafast thermocycling, has been spotlighted as a next-generation PCR system, overcoming key limitations of conventional PCR such as reliance on sophisticated instruments, high energy consumption and prolonged amplification time\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Recently, researchers have developed several photonic PCR systems based on Au film\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, colloidal nanoparticles (NPs)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, two-dimensional (2D) materials\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and carbon black\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Although these systems significantly shorten thermocycling time and advance PCR toward POCT, some limitations remain. On the one hand, high-cost lasers were required in some systems to match narrow plasmonic resonance band. On the other hand, large temperature difference may appear in planar heating-type plasmonic thermal cycler with large reaction volume, potentially compromising denaturation efficiency and promoting undesired primer-dimer formation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Moreover, several methods have been adopted for nucleic acid detection in these systems such as gel electrophoresis\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, fluorescent\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34 CR35 CR36\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and UV-Vis spectroscopy\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Fluorescence detection, while highly sensitive, is prone to interference from bubbles and chamber fog\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, requires costly equipment\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and is susceptible to photobleaching. Similarly, gel electrophoresis and UV-Vis spectroscopy require additional post processing or expensive equipment which would length the total assay time and conceal the advantage of rapid thermocycling. Therefore, a volumetric heating-type broadband absorption material could be considered to reduce the cost of light source and improve temperature uniformity. Additionally, lateral flow paper strip could also be considered to detect amplified products in photonic PCR for POCT, which would significantly shorten the total assay time, strengthen the advantages of rapid thermocycling and simplify the workflow.\u003c/p\u003e \u003cp\u003eHere, we developed a rapid plasmonic photothermal RT-PCR system for influenza A diagnosis, integrating plasmonic thermocycling and visual detection on single PCRstrip. BAPF was synthesized via in situ growth of Au NPs on porous fabric for efficient plasmonic heating. BAPF served as both a PCR solution container and a photothermal converter. Arising from densely packed, variably sized and randomly distributed plasmonic NPs, BAPF enabling strong broadband absorption in the visible-NIR range and rapid photothermal heating. Therefore, a simple and low-cost white light emitting diode (LED) on/off system was sufficient for the rapid thermal cycling for RT-PCR, while amplified products were visually detected on an immune-strip. The total assay of the PCRstrip was completed within 15 min with a limit of detection (LOD) of 1 copy/\u0026micro;L. The proposed photothermal RT-PCR device was further validated by testing nasopharyngeal swab samples collected from 61 influenza A and 19 non-influenza A patients, demonstrating a sensitivity of 98.4% and specificity of 94.7%. This fast, simple, accurate and cost-effective device may be a promising candidate for POCT and even for general population to use at home.\u003c/p\u003e"},{"header":"2. Experiment","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and methods\u003c/h2\u003e \u003cp\u003eTetrachloroaurate (III) trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e), trisodium citrate (TSC), thionine, (3-Aminopropyl) trimethoxysilane (APTMS), sodium silicate, bovine serum albumin (BSA), Triton X-100 and sucrose were purchased from Sigma (St Louis, MO, USA). Tri(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, 1 M, pH 8.5), DNA ladder, agarose, λ-DNA, 2 M TAE buffer, streptavidin (SA) and BSA-biotin were provided by Sangon Biotech Co., Ltd (Shanghai, China). 6-FAM monoclonal antibody (10 mg/ml) was obtained from OTwo Biotech (Guangzhou, China). RNA/RNAse free water was purchased from Tiangen (Beijing, China). DL1,000 DNA Marker, TaKaRa Z-Taq™ and One Step PrimeScript RT-qPCR Mix (2X) were received from Takara Biotech. Primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China) (Table S2). Influenza A RNA solution was gained from HZBio (Wuhan, China). STM32F407 microcontroller, white LED and LED driver were purchased from Alientek electronic Co., Ltd (Shenzhen, China), Philips and Telesky electronic Co., Ltd (Shenzhen, China), respectively. MAX31855, cooling fan, TFT LCD touch screen, Lithium battery and relay were gained from Risym electronic Co., Ltd. (Shenzhen, China).\u003c/p\u003e \u003cp\u003eGlass fiber (GF-08), sample pad, conjugate pad, absorption pad and backing pad were obtained from Jie Ning Bio Co. Ltd. (Shanghai, China). The nitrocellulose (NC) membrane was received from Whatman-GE Healthcare Company (UK). PET high-temperature tape and Kapton tape were bought from Taobao. Type-K thermocouple was obtained from KAIPUSEN (Jiangsu, China).\u003c/p\u003e \u003cp\u003eThe thermal images of BAPF were measured by an infrared thermographic camera (ETS320, FLIR). The morphology of BAPF was characterized by scanning electron microscopy (SEM; Zeiss, Ultra Plus). The absorption spectra of BAPF were measured with a UV-1900i UV-Vis spectrophotometer (Shimadzu, Japan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of BAPF\u003c/h3\u003e\n\u003cp\u003eGF-08 was cut into 25 mm × 25 mm and washed twice with deionized water. After drying, glass fiber (GF) was immersed into a mixture of 1 ml of 1% (w/v) HAuCl\u003csub\u003e4\u003c/sub\u003e and 1 ml of 100 mg/ml sodium citrate for 1 h. Washed away excess Au NPs, the GF color turned from white to black, which indicated the successful preparation of BAPF. Then, BAPF was immersed in 2% (v/v) APTMS in ethanol overnight for silanization. Washed by ethanol and water thoroughly, BAPF was treated with aqueous sodium silicate (0.054 wt%, pH 10 adjusted by 0.1 M NaOH) at 95°C for 1 h. Finally, BAPF was taken out, washed and dried. Finally, BAPF was cut into 5 mm × 5 mm and stored for further use.\u003c/p\u003e\n\u003ch3\u003ePreparation of PCR reaction mixture\u003c/h3\u003e\n\u003cp\u003eThe PCR reaction mixture of λ-DNA (total volume of 10 µL) consisted of 0.75 U TaKaRa Z-Taq, 0.3 µM of forward primer, 0.3 µM of reverse primer, 1 µL 10× Z-Taq Buffer (Mg\u003csup\u003e2+\u003c/sup\u003e Plus, 30 mM), 0.8 µL dNTPs, 0.5 µL 10 mg/ml BSA, 0.5 µL λ-DNA and certain amount of RNase-free water. RNA/RNAse free water was used as the template in negative control.\u003c/p\u003e \u003cp\u003eThe RT-PCR reaction mixture of influenza A RNA (total volume of 10 µL) consisted of 5 µL of PrimeScript 1 Step Enzyme Mix, 0.4 µM of forward primer, 0.4 µM of reverse primer, 1 µL of influenza A RNA and certain amount of RNase-free water. RNA/RNAse free water was used as the template in negative control.\u003c/p\u003e\n\u003ch3\u003eInhibitory effect of fabrics\u003c/h3\u003e\n\u003cp\u003eFabrics were cut into 5 mm × 5 mm squares and individually placed into PCR tubes containing 10 µL of PCR reaction mixture with λ-DNA templates. Each fabric was completely immersed in the solution and subjected to amplification in a conventional benchtop thermocycler. Subsequently, the amplified PCR products were analyzed by gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003ePCR amplification protocols in Benchtop instruments\u003c/h3\u003e\n\u003cp\u003eConventional PCR was carried out in an Applied Biosystems SimpliAmp thermal cycler (Thermo Fisher Scientific) following the protocols: 40 cycles of 98°C for 1 s and 60°C for 2 s. Conventional RT-PCR was conducted with following cycling conditions: 52°C for 5 min, 95°C for 10 s and 40 cycles of 95°C for 5 s and 60°C for 30 s. Quantitative reverse transcription PCR (RT-qPCR) was performed using the Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific). For reference RT-qPCR, samples with cycle threshold (C\u003csub\u003et\u003c/sub\u003e) values above 35 were classified as negative.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction from clinical samples\u003c/h2\u003e \u003cp\u003eClinical nasopharyngeal swab samples from influenza A patients were collected by Nanjing First Hospital and preserved in viral transport medium. Viral nucleic acids were extracted from 2 mL of viral preservation solution using an automatic nucleic acid extraction system (SSNP-2000B, Jiangsu Bioperfectus Technologies Co., Ltd.) with corresponding viral nucleic acid extraction kit (SDK60104). The extraction procedure, conducted in a Biosafety Level 2 (BSL-2) laboratory, consisted of 6 sequential steps: lysis, mixing, washing 1,2, elution and release, with a total processing time of approximately 45 min. The extracted RNA samples were subsequently analyzed by RT-qPCR to determine C\u003csub\u003et\u003c/sub\u003e values (Table S4) using following thermal cycling protocol: reverse transcription at 52°C for 5 min, initial denaturation at 95°C for 10 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. All RNA samples were stored at -80°C for subsequent analysis. This study received ethical approval from the Ethics Review Committee at Nanjing First Hospital (KY20230807-01-KS-01).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGel electrophoresis analysis\u003c/h3\u003e\n\u003cp\u003ePCR products were electrophoretically separated on a 2% (w/v) agarose gel containing 1× GelRed. Electrophoresis was performed at a constant voltage of 125 V for 35 min in 0.5× TAE (Tris/Acetic/EDTA) running buffer. Gel images were recorded by a GenoSens2150 Imaging system (CLINX Science Instruments, China).\u003c/p\u003e\n\u003ch3\u003ePreparation of the nucleic acid detection strip\u003c/h3\u003e\n\u003cp\u003eThe nucleic acid detection strip was composed of sample pad, conjugate pad, NC membrane, absorbent pad and backing pad. Sample pad, conjugate pad, NC membrane and absorbent pad were fixed onto the backing pad in order. During the assembly process, the ends of each part overlapped each other with about 2 mm to ensure continuous fluid flow by capillary effect. 6-FAM monoclonal antibody (1 mg/ml) and BSA-biotin (1 mg/ml) were fixed on the T line and C line, respectively. Finally, the assembled card was dried at 37°C for 1 h and subsequently cut into 3-mm wide test strips using a programmable cutting machine. The prepared strips were then stored in a desiccator for future use.\u003c/p\u003e \u003cp\u003e1 ml of 35 nm Au NPs was centrifuged at 7000 rpm for 10 min and resuspended in 0.5 ml of deionized water. Then, 8 µL of SA solution (1 mg/ml) was added and shaken gently at room temperature for 30 min. Next, 50 µL of BSA solution (10% w/v) was added and incubated for 2 h to block nonspecific binding sites. Then, the conjugated Au NPs was centrifuged 7000 rpm for 10 min and resolved in 50 µL of resuspending solution (1% BSA, 15% sucrose and 0.2% Triton X-100 in 20 mM Tris-HCl). Finally, Au NP/SA nanotag was impregnated onto the conjugated pad of paper strip and dried.\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003ch2\u003ePrinciple of the PCRstrip\u003c/h2\u003e\u003cp\u003eThe working principle of the PCRstrip is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, where nucleic acid amplification and detection are integrated into a handheld device. The BAPF containing the PCR reaction mixture was mounted at the front of the PCRstrip to enable ultrafast photothermal cycling. When illuminated by a white LED, photons excited plasmonic electrons on BAPF, generating hot electrons within 100 fs. These hot electrons, with low heat capacity, rapidly raised the temperature to several thousand degrees Kelvin, heating the surrounding PCR solution effectively. A microcontroller was employed to remotely control the LED via pulse-width modulation (PWM), precisely regulating irradiation intensity, duration and cooling cycles. Compared with laser-based systems, white LED consumed significantly less power and reduced the device cost, making it ideal for POCT.\u003c/p\u003e\u003cp\u003eAfter photothermal amplification, PCR products were released and flowed onto the paper strip for visual detection. The liquid migrated through the sample pad, conjugated pad, T line and C line. Target nucleic acids, amplified with end-labeled primers, bound to SA-functionalized Au nanotags (SA-Au nanotags), forming SA-Au nanotags-DNA complexes. At the T line, anti-FAM antibodies captured 6-FAM-labeled primers, producing a visible SA-Au nanotag-DNA-anti-6-FAM complex. In the absence of amplification products, Au nanotags were captured only on the C line. Therefore, the presence of target nucleic acid can be analyzed by observing the T line color. The entire process, including RT-PCR and visual detection, was completed within 15 min, facilitating rapid and accessible large-scale screening for POCT during pandemics.\u003c/p\u003e\u003ch2\u003eCharacterization of BAPF\u003c/h2\u003e\u003cp\u003eThe BAPF functioned as both a stable PCR solution container with high surface area and an efficient photothermal material. BAPF was prepared by in situ synthesis of Au NPs on GF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In this process, HAuCl\u003csub\u003e4\u003c/sub\u003e and TSC would infiltrate the interspace of GF via capillary effect, reducing Au\u003csup\u003e3+\u003c/sup\u003e to Au NPs on GF over time. The BAPF exhibited strong broadband light absorption in the visible range, enabling rapid photothermal conversion and efficient heat transfer to the surrounding PCR solution. Meanwhile, GF exhibited a three-dimensional multi-interspace structure with pores at micrometer scale, which provided favorable condition for efficient accommodation and uniform distribution of the PCR reaction solution (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Moreover, the highly porous structure facilitated internal mass transfer required for an effective reaction. Therefore, BAPF functioned as volumetric heating source rather than planar heating source. Since the solution was contained in the membrane, the surface area for heating per solution volume was higher than that in the PCR tube\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Plasmonic heating could effectively heat the whole PCR solution and the heating rate was increased greatly.\u003c/p\u003e\u003cp\u003eIn fact, the photothermal performance of BAPF strongly depended on the loading amount of Au NPs. Therefore, the reaction time (10, 20, 30, 45, 60 and 120 min) for BAPF was firstly optimized. As the reaction time increased, the loading amount of Au NPs increased, turning the BAPF from light gray to black. SEM images in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb confirmed that the loading amount of Au NPs on GF increased initially and saturated over 60 min. The optical absorbance spectra of six BAPFs were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. All BAPFs exhibited broad and flat absorbance in the visible light range may arising from the hybridization localized surface plasmon (LSP) modes from nanometer-sized Au nanoclusters with randomized sizes and positions, resulting in disordered plasmonic broadband absorption performance\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e–\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The absorbance of BAPF increased with reaction time as more Au NPs formed and densely packed on the fabric, however, saturated over 60 min. Since the BAPF was a strong absorber of light over full spectrum of visible light, a cost-effective white LED was used for photothermal conversion. Upon illumination, the temperature of BAPF increased rapidly, heating the entire sample uniformly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). In contrast, the pristine GF barely sensed the white light, resulting in unnoticeable temperature changes under identical condition. The marked contrast further confirmed the thermoplasmonic mechanism underlying the observed thermal response in BAPF. To assess the photothermal effect dependence on the Au NPs loading amount, six BAPF samples synthesized with different reaction time were illuminated by the white LED for a certain time and cooled (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The heating rate increased initially and saturated over 60 min which was in coincidence with optical absorbance. Therefore, the reaction time of BAPF was optimized at 60 min for subsequent experiments. To further evaluate the photothermal responsiveness of BAPF, temperature profiles of BAPF illuminated by LED modulated by different PWM duty cycles were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg. These results demonstrated precise control over heating dynamics achievable with the BAPF, making it an ideal candidate for ultrafast photothermal PCR applications.\u003c/p\u003e\u003cp\u003eInhibitory effect was also a significant factor to be considered. The inhibitory effect of fabrics on PCR was investigated and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh. GF shown barely no adverse effect on PCR which made it a good candidate for containing PCR solution. However, BAPF shown great inhibitory effect on PCR arising from the strong adsorption of polymerase on Au NPs\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Therefore, a hermetic silica shell was coated on Au NPs to eliminate PCR inhibition arising from polymerase adsorption. To ensure that the silica shell is pinhole-free, SERS spectra of thionine were measured on BAPF and silica coated BAPF, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, the overall shape of 1350 to 1450 cm\u003csup\u003e− 1\u003c/sup\u003e region in the spectra of thionine on silica coated BAPF was similar with that in the ordinary Raman spectra. The intensity of 1120 cm\u003csup\u003e− 1\u003c/sup\u003e was relatively low in the ordinary Raman spectra and the spectra on silica coated BAPF, whereas high in the SERS spectra on BAPF without silica shell. Above differences were in consisted with that in reported results, proving the shell was pinhole-free\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Obviously in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, BAPF passivated with silica shown negligible inhibitory effect on PCR owing to the imporous silica coating. In addition, the silica shell offered Au NPs with thermal stability against the reshaping of the nanoparticle (Figure S2). Moreover, the optical image of large-scale-manufactured-BAPF (110 mm × 110 mm) was shown in Figure S3, which can be divided into 484 individual 5 mm × 5 mm units. Consistent photothermal performance was observed in six batches of BAPF (Figure S4), proving the excellent reproducibility and process stability. Therefore, BAPF as an excellent plasmonic photothermal material can be reliably produced in a high-throughput scale.\u003c/p\u003e\u003ch2\u003eNumerical simulation\u003c/h2\u003e\u003cp\u003eThe temperature difference is a critical factor affecting the accuracy and reproducibility of plasmonic photothermal nucleic acid amplification\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. As a proof-of-concept, a numerical simulation employing finite element method (FEM) was performed in COMSOL Multiphysics 5.6 to compare the temperature distribution of photothermal heating of solution in planar plasmonic Au substrate and BAPF. The model parameters of planar plasmonic Au substrate were adopted from a reported paper\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, consisting of a 120 nm-thick Au membrane. The detailed model structure was shown in Figure S5 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. For the model of BAPF, the structure was simplified as orthogonal stacked layers of Au-coated SiO\u003csub\u003e2\u003c/sub\u003e fiber, with each fiber having a diameter of 20 µm and a center-to-center distance of 50 µm between adjacent layer. The detailed structural design and geometric parameters were shown in Figure S6 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Two physics Electromagnetic Waves and Heat Transfer in Solids and Fluids were coupled together to calculate the resulting temperature illuminated by a light beam. Numerical simulation results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. When the Au film was used as the heating element, heat was transferred from the bottom upward, leading to reduced heating uniformity. In contrast, BAPF exhibited a three-dimensional multi-interspace structure with micrometer-scale pores that allowed volumetric heating, resulting in even heat distribution throughout the reaction mixture. As obviously illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the temperature difference in BAPF exhibited the maximum difference 0.6°C, while that was 2.5°C in Au film. Therefore, the homogeneity of heating is greatly improved in BAPF, which is conducive to preventing the decline of denaturation efficiency and the undesired primer-dimer formation.\u003c/p\u003e\u003cp\u003eMoreover, the use of BAPF as a solution container increased the surface area-to-volume ratio for heating and cooling\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The numerical simulation showed that the solution in BAPF took 1.7 s to reach 98°C from 20°C, whereas that in the Au membrane took 2.5 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Similarly cooling of solution in BAPF took 170 s to reach 20°C from 98°C, whereas that in the Au membrane took 330 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). These results demonstrated that BAPF enabled volumetric heating, facilitating significantly faster thermocycling while ensuring uniform thermal distribution across the entire sample volume with minimal thermal gradients.\u003c/p\u003e\u003ch2\u003ePhotothermal PCR in BAPF\u003c/h2\u003e\u003cp\u003eThe photothermal PCR performance of BAPF under white LED illumination was firstly investigated using λ-DNA as the template. A piece of BAPF containing 10 µL of PCR solution was sealed by two pieces of transparent PET tape to prevent evaporation during thermocycling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Noticeably, the PET tape was incubated by 10% (w/v) BSA in advance to prevent nonspecific adsorption of polymerase. A thin thermocouple was attached tightly onto BAPF to measure the temperature for closed-loop control. Meanwhile, signals related to real-time temperature were transmitted to a microcontroller through a MAX31855 thermocouple module. Then, the sealed BAPF was mounted right above a white LED for thermocycling. STM32F407 with embedded code controlled the LED irradiation intensity and fan on/off through I/O port, enabling precisely controlled thermocycling. Stable thermocycling results of 40 cycle PCR within 373 s were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, with the heating rate of 11.875°C/s and cooling rate of 8.137°C/s. A typical temperature profile of a single thermal cycle was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, the heating stage from 60°C to 98°C was realized by white LED illumination and the cooling stage was accomplished by white LED off and fan on. 100 pieces of BAPF were randomly selected to perform 40 cycles with annealing/extension at 60°C and denaturation at 98°C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The temperature fluctuations were minimal, with standard deviations of 1.35°C at 98°C and 0.3°C at 60°C, proving the high consistency and reliability of photothermal temperature control achieved by BAPF. Henceforth, the BAPF can be used for subsequent nucleic acid amplification applications.\u003c/p\u003e\u003cp\u003eAfter photothermal PCR, the amplification products were extracted by centrifugation for gel electrophoresis analysis. The electrophoretic band of λ-DNA (98 bp, Table S3) amplified by benchtop device and BAPF were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, demonstrating matching band and comparable amplification performance. The fluorescence intensity of each electrophoretic band was quantified using ImageJ software. Compare to the PCR products obtained from a conventional PCR thermocycler, the photothermal amplification yield exceeded 85%. In addition, the photothermal PCR exhibited reproducible DNA amplification results, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee. PCR products from eight independent photothermal PCR tests shown similar gel electrophoretic band intensity. Gel electrophoresis analysis results of λ-DNA dilutions (0-0.1 ng/µL) after photothermal PCR were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef. The band intensity gradually decreased as the λ-DNA concentration reduced from 0.1 ng/µL to 0, with a visual detection limit of approximately 0.1 pg/µL. These results demonstrated that DNA can be efficiently amplified in BAPF by photothermal PCR, confirming the feasibility of proposed strategy.\u003c/p\u003e\u003ch2\u003eFabrication and working principle of photothermal PCR chip\u003c/h2\u003e\u003cp\u003eThe photothermal PCR chip (107 mm × 44 mm × 11 mm) was developed by integrating the BAPF and lateral flow paper strip in a customized three-dimensional (3D) printing plastic housing for on-site rapid diagnosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The casing had an opening in the front and the other in the middle. The front opening was designed to expose BAPF for light illumination and fan cooling. The other opening was for visual observation of nucleic acid paper strip. BAPF was sealed by two pieces of PET tape and mounted in the front of the chamber. Subsequently, a lateral flow paper strip was fixed in the middle rear of the chamber. Importantly, a 2 mm overlap was kept between BAPF and conjugated pad to ensure amplified products released from BAPF flowed onto test strip through capillary force successfully. RT-PCR is currently the most sensitive and specific detection method of viral RNA\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. To assess whether this photothermal amplification strategy is also applicable to RNA targets, RT-PCR has also been carried out with influenza A RNA. The on/off duty of white LED was slightly modified to conduct thermocycling for RT-PCR. The RT-PCR protocol began with a constant temperature at 52°C for 3 min (\u0026lt; 1°C variation) for sufficient complementary DNA (cDNA). Then, 40 cycles of PCR amplification were conducted between 95°C and 60°C for denaturation and annealing/extension (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The entire RT-PCR can be finished within 10 min in proposed photothermal PCR chip. M1 gene (123 bp) of influenza A RNA was used as a positive control to demonstrate the capability of proposed photothermal RT-PCR chip (Table S3). The photothermal RT-PCR products of various concentrations of Influenza A RNA were analyzed by gel electrophoresis, with fluorescence intensity correlating with RNA concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eFor visual detection of Influenza A RNA, the PET tape was punctured after photothermal RT-PCR to release amplified products onto the lateral flow paper strip (Figure S7). Consequently, PCR products labeled with biotin and 6-FAM at opposite ends would form complexes with SA-Au nanotags (Table S2). Complexes were immobilized on the T line due to the affinity of 6-FAM and anti-6-FAM antibody. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee shown the lateral flow paper strip detection results of different concentrations of influenza A RNA after ultrafast photothermal RT-PCR. The color intensity of T line varied with the RNA concentration, achieving a visual detection limit of approximately 1 copy/µL. Additionally, test strips for six repetitions of influenza A RNA photothermal RT-PCR products demonstrated excellent reproducibility and accuracy (Figure S8). Therefore, this BAPF-based photothermal chip was also applicable for RNA targets with high sensitivity and stability.\u003c/p\u003e\u003ch2\u003eClinical diagnosis of nasopharyngeal swab samples on POCT device\u003c/h2\u003e\u003cp\u003eTo enable POCT applications, an integrated opto-mechatronic system was developed to enhance the portability and functionally of the device (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The POCT device measured 107 mm × 96 mm × 103 mm and weighed 437 g, with all optoelectronic components encapsulated in a custom 3D-printed housing for automatic PCR operation. The system consisted of several key components that work in concert for precise and efficient thermocycling. A white LED, serving as the photothermal excitation source, was mounted on a cooper radiator equipped with a cooling fan to dissipate heat generated during operation. Above the BAPF, another 12 V fan was installed to accelerated the cooling rate during thermocycling process, enabling rapid temperature transitions between heating and cooling phases. The microcontroller unit (MCU), LED driver and relay were integrated on a printed circuit board (PCB). As illustrated in the control schematic, the MCU implemented a digital proportional-integral-derivative (PID) temperature control algorithm to precisely regulate the thermocycling process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The MCU monitored temperature via a type-K thermocouple, continuously calculated the error between the preset and measured values, and dynamically adjusted output variables through PWM to maintain the desired temperature profile. This closed-loop control mechanism ensured high accuracy and reproducibility in temperature regulation. For user interaction, a TFT LCD touch panel provided an intuitive graphical interface, allowing users to input PCR parameters (e.g., cycle number, temperature settings) and display real-time temperature curves during operation (Figure S9). For remote operation, a mobile application was developed to communicate wirelessly with the instrument via Bluetooth. The application, installed on a smartphone, featured a user-friendly interface for inputting amplification parameters and real-time display of amplification progress (Figure S10). The whole device was powered by a rechargeable lithium battery, making it highly portable and suitable for POCT applications. To initiate operation, the user simply turned on the power button, inserted the photothermal chip preloaded with PCR solution, and input PCR parameters via the touch panel or mobile app. Once started, the system automatically executed ultrafast photothermal thermocycling, with the MCU coordinating the LED, fans and sensors to ensure optimal performance.\u003c/p\u003e\u003cp\u003eTo validate the clinical applicability of our system, we conducted additional tests using the palm-size plasmonic photothermal PCR instrument. Nasopharyngeal swab samples collected from patients infected with influenza A virus were analyzed using the customized prototype device. A total of 80 human saliva specimens (61 positives and 19 negatives) were tested. The C\u003csub\u003et\u003c/sub\u003e values of all samples were confirmed in advance using a conventional benchtop RT-qPCR after the extraction of viral RNA from the swabs (Table S4 and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The whole workflow, illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, included sample collection, RNA extraction, sample loading, photothermal PCR and detection. The photothermal RT-PCR protocol included an initial reverse transcription at 52°C for 3 min, followed by 40 amplification cycles of denaturation at 95°C and annealing/extension at 60°C. Upon completion of the RT-PCR final cycle, amplicons were released and the results could be visually assessed using lateral flow strips by naked eyes. The resulting images of lateral flow paper strips were shown in Figure S11 and the T line intensity of all samples were mapped in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee. Using proposed photothermal RT-PCR device, 60 of 61 positive samples were identified, while all the negative controls except one were identified correctly. Noticeably, positive samples exhibited significantly higher levels of T line intensity than negative samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). The clinical performance of proposed photothermal RT-PCR showed a diagnostic agreement of 96.5% compared to the conventional RT-qPCR and demonstrated a clinical sensitivity and specificity of 98.4% and 94.7%, respectively. To further compare the performance of the POC device with conventional RT-qPCR, normalized C\u003csub\u003et\u003c/sub\u003e values and normalized T line intensity were plotted and linear fitted. The Pearson correlation coefficient (r) was − 0.93, which proved the existence of a correlation between the conventional RT-qPCR and proposed method (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Overall, proposed device had the potential to conduct influenza A molecular diagnosis in clinical situation with a significantly short operation time of 15 min. Obviously, nucleic acid testing of other diseases can also be conducted on proposed device after primers design and amplification protocol optimization. This device shown remarkable features, including: (1) the assay was based on well-established RT-PCR rather than isothermal amplification test. (2) the greatly shortened analysis time was conducive to on-site diagnosis. (3) mobile and professional-free operation alleviated the pressure of laboratory testing during pandemic. (4) ultralow cost was beneficial to POC testing (Table S5). (5) naked eye detection greatly saved detection time. According to these prominent advantages, our platform is of great potential for personal timely molecular diagnosis of various diseases in POCT.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, a simple, cost-effective and robust POC photothermal RT-PCR device has been developed for personal pathogen diagnosis in POCT. The device relied on a small photothermal chip and a portable photothermal instrument, which accomplished rapid amplification and naked-eye detection without additional expensive PCR infrastructure and power supply. Plasmonic photothermal nanostructure and lateral flow paper strip were integrated in single PCRstrip for ultrafast nucleic acid amplification and visual detection. BAPF which exhibited strong and broadband absorption in entire visible range produced ultrafast nanoplasmonic heating resulting from strong light-to-heat conversion of white LED. Programmable and automatical thermocycling was completed in a customized compact prototype machine within 15 min (10 min for RT-PCR and 5 min for detection). The clinical diagnostic performance of proposed device was validated with sensitivity and specificity of 98.4% and 94.7%. Proposed PCRstrip combines advantages of low cost, rapid amplification time and compact size, making it a promising tool for POC molecular diagnosis in resource-limited or personal situations in against of pandemic and for the distinction diagnosis with other respiratory viruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge the financial supports from National Natural Science Foundation of China (82361138570, 82402755), Shenzhen Science and Technology Plan Project (JCYJ20230807114610021) and Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (KYCX22_0246).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJung WE, Han J, Choi J-W, Ahn CH (2015) Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies. 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ACS Nano 14:5268\u0026ndash;5277\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"plasmonic photothermal, photonic PCR, broadband absorption, POCT","lastPublishedDoi":"10.21203/rs.3.rs-6336864/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6336864/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe diagnosis of respiratory viral infection via reverse transcription-polymerase chain reaction (RT-PCR) is typically conducted in centralized laboratories using bulky equipment costing 1\u0026ndash;2 h. To prevent the spread of infectious diseases, there is an urgent need for ultrafast and accessible molecular diagnostic tools for point-of-care testing (POCT). Here, we developed an ultrafast, POC molecular diagnostic PCRstrip capable of detecting influenza A RNA with high sensitivity (1 copy/\u0026micro;L) in just 15 min. Our system integrated reverse transcription, rapid thermocycling and visual detection at ultralow cost. Broadband absorption plasmonic fabric (BAPF) with excellent photothermal effect was fabricated for volumetric photothermal heating excited by a cost-effective white LED. After photothermal PCR, amplification products can be visually detected by a lateral flow paper strip. Clinical validation of 80 nasopharyngeal swab samples collected from patients suspected of influenza A infection demonstrated a clinical sensitivity of 98.4% and specificity of 94.7%. This fast, ultralow-cost and reliable molecular diagnosis strategy is conducive to POCT, offering an effective tool for non-trained personnel to detect and control infectious diseases individually.\u003c/p\u003e","manuscriptTitle":"Ultralow-cost personal PCRstrip with volumetric heating based on broadband absorption plasmonic fabric for POCT of pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 09:43:01","doi":"10.21203/rs.3.rs-6336864/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9e7f15e6-d293-4f35-9e74-88bbbc005aef","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47265112,"name":"Biological sciences/Biotechnology/Assay systems"},{"id":47265113,"name":"Biological sciences/Biological techniques/Sensors and probes/Surface plasmon resonance"},{"id":47265114,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures"},{"id":47265115,"name":"Physical sciences/Nanoscience and technology/Nanoscale devices/Nanophotonics and plasmonics"},{"id":47265116,"name":"Physical sciences/Engineering/Biomedical engineering"}],"tags":[],"updatedAt":"2025-05-05T09:43:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 09:43:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6336864","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6336864","identity":"rs-6336864","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}