A laser engraver (KB-4060) was bought from Liaocheng Keba Laser Equipment Co., Ltd. (Shandong, China). Fluorescence spectrum was acquired using a NanoDrop 3300 fluorescence spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Thin polycarbonate (PC) sheets (thickness: 1 mm) were purchased from Shanghai Chenchuang Plastic & Rubber Technology Co., Ltd. (Shanghai, China), sealing membranes were purchased from Rongxin Packaging Material Co., Ltd. (Shenzhen, China). Regenerated cellulose membrane filters with 0.2 μm bore diameter were purchased from CHMLAB group (Barcelona, Spain). The heater (XH-RP5050) was purchased from Jiangsu Xinghe Electronics Co., Ltd. (Jiangsu, China), andthe rotator from Jiangsu Xinkang Medical Equipment Co., Ltd. (Jiangsu, China). The UV Analyzer (ZF-7A) was purchased from Shanghai Qinke Analytical Instrument Co., Ltd. (Shanghai, China). Polydopamine was purchased from Sigma Aldrich (St. Louis，USA). Aseptic paraffin oil was purchased from Hengkang Medical (Hebei, China). LAMP kits containing detection reagent (Bst DNA polymerase and primer), reconstitution fluid (10x isothermal amplification buffer solution, dNTP mixture, 100mM MgSO4), colorimetric indicator (calcein, including Mn2+), a positive control (target bacteria DNA), and a negative control (non-objected bacteria DNA) were purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. (Guangdong, China). Biowest agarose and loading buffer were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). A fully-automatic gel-imaging analysis system (Shanghai, China) was used to test the target band. 100bp DNA marker and genomic DNA extraction kit were purchased from Takara (Shiga, Japan).
Bacteria preparation and DNA extraction
E.coli O157:H7, Salmonella spp., S. aureus, and V. parahaemolyticus were obtained from the Institute of Hygiene and Environmental Medicine (Tianjin, China). E. coli O157:H7, Salmonella spp., S. aureus, and V. parahaemolyticus were cultured overnight in 5 mL Lysogeny Broth (LB) (37℃, 200 rpm oscillation). gDNA was extracted from 1 mL of culture solution using the DNA purification kit. gDNA concentration and mass was determined by UV-visible spectrophotometer and NanoDropTM spectrophotometer. gDNA was then stored at -20℃ for future use.
Optimizing LAMP reaction conditions
To optimize the reaction temperature and time of LAMP on the microchip, the reaction temperature was set to 60℃, 65℃ and 70℃. The effect of temperature on the reaction was then determined by the resulting fluorescence intensity. This approach was used to determine the optimal reaction temperature. Optimal reaction time was determined in a similar manner. The reaction time was set to 15 min, 20 min, 25 min, 30 min, and 40 min, and optimal reaction time was also determined based on fluorescence intensity.
Testing calcein fluorescence
As previously reported mix 25 umol L-1 calcein with 300 umol L-1 manganese chloride to quench the fluorescence of calcein. Add the quenched calcein to the LAMP reagent, then shake the solution and observe it under UV radiation. Next, remove the solution and place it in a 65℃ water bath for 30 min, then observe the solution under UV radiation again. To test the fluorescence of calcein on paper, soak the paper in the quenched calcein solution and let it dry at room temperature. After drying, check the paper under UV radiation. Last, use the paper dipped in calcein to test the LAMP byproduct.
The portable microfluidics chip used for multichannel LAMP testing is made in two layers. The 40×40×1 mm PC board is composed of eight reaction chambers, each having a radius of 2.5 mm. Reaction chambers are connected to the center chamber, which has a radius of 8mm, by a 5 mm micro-channel that is 0.5 mm deep. The total volume of each reaction chamber and sample chamber is 10 μL and 100 μL, respectively. The hole at the cavity position is made by a computer-aided direct current engraving machine and the upper part of the PC plate is sealed by the sealing membrane. First, cut the cavity and the channel on the PC plate shown in Fig. 7a. Second, embed the regenerated cellulose paper plate with 0.2 μm pores with LAMP reagent stored in the reaction chamber in Fig. 7b. Third, attach the upper-part of the sealing membrane to the top of the PC plate as Fig. 7c. The device is then ready for amplifying various DNA templates from the sample as Fig. 7d.
Use the LAMP kit from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd. which contains primers for the target bacteria. Because there is no need for self-designed primers, the experimental workflow can be further simplified. To amplify and test multiple DNA templates with the device, soak each kind of paper in the reaction chamber with different primers, keep each reaction chamber containing the dry LAMP reagent. Then amplify the paper plate containing different target DNA primers and calcein. Inject the mixed solution containing template DNA of E. coli O157:H7, S. aureus, Salmonella spp., and V. parahaemolyticus into the sample chamber via the inlet on the upper layer of the sealing membrane. Then position the device and set the rotator’s velocity to 4000 rpm to uniformly push the sample solution into the reaction chamber via centrifugal force. After completing the rotation step, bring the sample solution to 10 µL in each reaction chamber, then place the device on the portable heater and perform the LAMP reaction at the optimal reaction temperature and time. Store the reagent with the paper plate before placing it in the cavity, doing so eliminates the need for steps involving sample and reagent injection, which is different from the complicated design of other technologies that rely on different rotating speeds.
On-microchip LAMP test
Prior to starting the reaction, place the paper plate containing quenched calcein into the reaction chamber. After the reaction is complete, the pyrophosphate ions and manganese ions combine to show the fluorescence signal of calcein under UV radiation. To verify the test, LAMP amplicons were subjected to AGE for 30 min, and then photographed under transparent UV radiation using the Bio-Rad Molecular Imager Gel Chemi Doc XR imaging system.
Sensitivity and Specificity testing
Test the sensitivity by performing a continuous dilution 10 times of the initial concentration of pathogen gDNA to determine the sensitivity of the microfluidic device’s visual inspection of LAMP amplicons. Use the UV-visible spectrophotometer to measure gDNA concentration by the following equation: DNA concentration = F×A260×molar absorption coefficient (ng μL -1), where F is the dilution ratio of the original DNA solution before measurement and A260 is the absorbency reading at 260 nm. The molar absorption coefficient of double-stranded DNA is 50 ng μL -1. Use only E. coli O157:H7 gDNA to evaluate the sensitivity of the device and verify the results by AGE for 30 min.
Use the microfluidic device to test the specificity of the LAMP test for gDNA at the lowest detectable concentration based on the sensitivity experiment. Use only the E. coli O157:H7 gDNA to evaluate the specificity of the device. Place the primers for E. coli 0157:H7, Salmonella spp., S. aureus, and V. parahaemolyticus in chambers 1-4, respectively. Inject the template DNA of E. coli O157:H7 into the central sample chamber and use chambers 5-8 as negative control chambers. Transfer the solution in the central sample chamber to the reaction chambers via centrifugal force, then heat the device for reaction on the heater at 65℃ for 30 minutes. Take 3 uL of the reaction solution to verify amplification by AGE after the reaction is complete.
Using real samples for on-chip analysis
Insert the paper coated with polydopamine into the central sample chamber to purify DNA from the degenerative milk solution. First, add the Salmonella spp. bacteria solution into the milk and incubate at 37℃ for 12 hours. Then heat the degenerative milk at 90℃ for 5 min to destroy bacterial cell walls. Next, incubate the solution at room temperature for 30 min to prepare the bacterial sample and polydopamine-coated paper for sufficient reaction. Lastly, apply centrifugal force to distribute the purified DNA solution to each reaction chamber, after loading the sample, conduct the on-chip LAMP reaction and the follow-up fluorescence detection. See Fig. 8 for the entire operation workflow.