Compact highly sensitive photothermal RT-LAMP chip for simultaneous multidisease detection

Abstract

Developing instant detection systems with disease diagnostic capabilities holds immense importance for remote or resource-limited areas. However, the task of creating these systems-which are simultaneously easy to operate, rapid in detection, and cost-effective-remains a challenge. In this study, we present a compact highly sensitive photothermal reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) chip (SPRC) designed for the detection of multiple diseases. The nucleic acid (NA) amplification on the chip is achieved through LAMP driven by either LED illumination or simple sunlight focusing. SPRC performs sample addition and amplification within a limited volume and autonomous enrichment of NA during the sample addition process, achieving a limit of detection (LOD) as low as 0.2 copies per microliter. Through 120 clinical samples, we achieved an accuracy of 95%, with a specificity exceeding 97.5%. Overall, SPRC has achieved promising progress in the application of point-of-care testing (POCT) by using light energy to simultaneously detect multiple diseases.

Fig. 1.. Schematic illustrations and working principle of sensitive photothermal RT-LAMP chip (SPRC).

(A) Structural explosion view of SPRC. (B) Heating methods using LED irradiation and sunlight focusing. (C) Workflow of SPRC. (D) Compact unit for easy use of SPRC. (E) Comparison of our work with conventional PCR methods. PES, polyethersulfone.

Fig. 2.. Au film photothermal and filter paper modification.

(A) Electromagnetic field distribution of the Au film with a thickness from 10 to 120 nm. (B) Highest temperatures of Au films with different thicknesses (10 to 150 nm) after being irradiated by LEDs of different powers (characterizing by current: 300 to 1500 mA for 3 min). (C) Heating rate and resistance change rate of a combination of the Au film with different sizes and planar condensers with different angles. (D) Partial enlarged images of sampling and amplification positions to show the working process. (E) Process of modifying chitosan oligosaccharides on the surface of Fusion5. (F) Capture efficiency of the DNA with the input amounts of 0.5, 1, 2, 4, and 8 ng. (G) Fluorescence images of the particle filtration for each filter layer. (H) Adsorption efficiency of DNA samples with concentrations of 1, 100, and 10 pM on the surface of Fusion5. a.u., arbitrary units.

Fig. 3.. On-chip elution and performance of SPRC.

(A) Time required for samples to flow through filter membranes with different pore sizes. (B) Effects of different volumes of DI water flushing on amplification curves. (C) Flow rate and DNA capture efficiency of filter membranes with different pore sizes. (D) Uniformity of solution flowing into each chamber. (E) Diffusion time of NA from Fusion5 to solution. (F) LAMP amplification curve for different contact times between Fusion5 and the gel reagents. (G) The sealing effect of paraffin particles on the reaction system. (H) Effect of agarose and paraffin on LAMP. Fluorescence images were captured by a camera, gray-scale calculations were performed to obtain fluorescence values, and the detection threshold was set to five times the SD of the blank. (I) Detection limit of HBV and HCV.

Fig. 4.. On-chip temperature sensor and chip performance.

(A) Thermal imaging of Au-plated glass under the illumination form five light sources of 3 W. (B) Numerical simulation of a single chamber heating process. (C) Heating performance under the input voltage of an amplitude no more than 5 V. (D) Temperature variations of the test solution within five chambers during LAMP reaction. (E) Schematic diagram of SPRC assembly and storage. (F) Time threshold (T_t) value of the SPRC after the storage for 4 weeks. (G) Explosion view of the compact device. (H) Operating principle of the compact device.

Fig. 5.. Testing of Serum samples with SPRC for various diseases diagnoses.

(A) Clinical trial protocol. A total of 320 tests were analyzed by SPRC and conventional reverse transcription PCR (RT-PCR), including 30 patients with hepatitis B, 15 patients with hepatitis C, 15 patients with influenza A, 10 patients with AIDS, and 10 healthy individuals. (B) Repeatability test of HBV and HCV based on the SPRC (****P < 0.0001). (C) Normalized fluorescence signal levels in 320 tests. The four vertical color blocks represent blood test results from the same volunteer. (D) Fluorescence value of most positive samples (+) is significantly higher than that of negative (−) (**P < 0.01 and ****P < 0.0001). (E) Evaluation of the consistency between SPRC and RT-PCR analyses showed positive correlations (Pearson’s r values: r_HBV = 0.82; r_HCV = 0.86; r_IAV = 0.80; r_HIV = 0.78). (F) Receiver operating characteristic (ROC) curves. The dashed line represents the threshold estimated by ROC analysis. The area under the curve of all pathogens is close to 1.

Fig. 6.. Detecting nasopharyngeal swab samples with SPRC.

(A) Standardized fluorescence levels of 20 patients with adenovirus (ADV) and 20 patients with COVID-19 (N: nucleocapsid gene, E: envelope gene, O: open reading frame 1a gene). (B) Fluorescence detection results of ADV showing significant differences between positive and negative samples. The threshold for positive results was set at five times the SD of negative results (****P < 0.0001). (C) Comparison of detection results from SPRC and RT-PCR Ct values, with a Pearson’s r value of 0.82. (D) Fluorescence values of patient with COVID-19 were significantly higher than those of the negative group. (E) Evaluation of analytical concordance between SPRC and RT-PCR. The results showed a positive correlation (Pearson’s r values: r_N = 0.84; r_E = 0.77; r_O = 0.71). (F) Appearance of sunlight-driven device. (G) Using sunlight-driven devices for the detection of ADV, successfully detecting 28 of 30 samples (93.33%). (H) Fluorescence detection results of ADV under sunlight driving. ****P < 0.0001. (I) Comparison of sunlight detection results with RT-PCR Ct values.