Naked eye detection of multiple tumor-related mRNAs from patients with photonic-crystal micropattern supported dual-modal upconversion bioprobes† †Electronic supplementary information (ESI) available: Experimental details and supplementary results. See DOI: 10.1039/c6sc03401b Click here for additional data file.

We have designed a biochip-based mRNA detection device by combining a hydrophilic–hydrophobic micropattern with upconversion luminescence (UCL) probes.


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Sample characterization. The morphology of the PEI-coated UCNPs was characterized by a JEM-2100 transmission electron microscope (JEOL) with an accelerating voltage of 200 kV.
Zeta potential measurements were performed using a Malvern Zetasizer Nano ZS system. The phase of nanocrystals was carried out using a D8 Advance X-ray diffractometer (Bruker) with a Cu-K radiation (λ = 1.5406 Å). UV-vis transmission spectra were obtained on a UV-2550 scanning spectrophotometer (Shimadzu). Luminescence spectra were recorded on a Hitachi F-4600 fluorescence spectrometer equipped with a 980 nm CW laser and an 808 nm laser (Kai Site Electronic Technology Co., Ltd.). Contact angles were captured by OCA20 instrument (Dataphysics, Germany) at 25 °C. Deionized water (MilliQ, 18.5 MΩ cm) was employed as the source for the CA measurement. The full image of the PC microchip was captured by digital camera 60D (Canon Co., Ltd). The luminescence images were captured by an unmodified camera phone. 3 . In a typical preparation, a kind of lanthanide oxide was added into the appropriate amount of water to form a slurry, then slighted excessive trifluoroacetic acid was added into the forementioned slurry under vigorously stirring. The whole solution was kept refluxing under 140 °C until a transparent solution was formed. The resulting solution was transferred into an evaporating basin after a filtered step. With the water evaporated, RE(CF 3 COO) 3 powders were obtained. centrifugation. Finally, the PEI-UCNPs were dispersed in ultrapure water after washing three times with ethanol and water (v/v = 1:1).

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Fabrication of the PC dots-based substrate. The PC dots-based substrate with a hydrophilichydrophobic micropattern was successfully fabricated through a solvent evaporation method. In details, PDMS and the curing agent (m/m = 10:1) were first blended, followed by vigorously stirring for 30 min and then was treated under vacuum until there was no bubble any more. Then the mixture of PDMS and curing agent was spin-coated on the clean cover glass, and then the PDMS coated glass was cured in an oven at 60 °C overnight to form a transparent and hydrophobic PDMS substrate. Next, 2 μL of monodispersed latex spheres (polystyrene, PS) suspension was dropped vertically on the PDMS substrate, and then was heated on the heating plate at 40 °C and maintained at this temperature until the PCs droplet was dried. Finally, these prepared substrates were placed in a dry condition for further use.
Preparation of mRNA detection device and detection of mRNA. 8 mg of EDC and 12 mg of NHS was dissolved in 1 mL of MES buffer to act as the activator, and then 2 μL of the activator was dropped to each PC dot and reacted for 10 min, followed by washing with ultrapure water. After drying, 2 μL of as-prepared mRNA probes was added to each PC dot and reacted at 30 °C for 30 min. Then, the PC dots were washed with PB buffer. For the S6 detection of mRNA, two kinds of experimental methods were carried out. In the first experimental method, the target mRNA was added first, and then the GO sheets were added. [4] In details, each PC dot was added 1 μL of targets containing TK1 mRNA and C-myc mRNA (TK1 perfectly matched target: 5'-AAGTATGCCAAAGACACTCGC-3', C-myc perfectly matched target: 5'-CCTCAACGTTAGCTTCACCAA-3'). After reacting at 25 °C for 2 h, the device was washed with PB buffer for one time. Then, 1 μL of GO (0.3 mg mL 1 ) was dropped to each PC dot and reacted at 25 °C for 30 min to combine with the probes that without hybridization. Finally, the device was washed with ultrapure water for three times to remove the excess GO. A 980 nm CW laser and an 808 nm CW laser were used to irradiate the detection device and the luminescence images were captured by an unmodified camera phone. In the second experimental method, the GO sheets were added first to quench the luminescence of the mRNA probes, and then the target mRNA was added to restore the luminescence. [5,6] In details, each PC dot was added 1 μL of GO (0.3 mg mL 1 ) and reacted at 25 °C for 30 min to combine with the probes. Then, the PC dots were washed with ultrapure water for three times to remove excess GO. Finally, 1 μL of targets containing TK1 mRNA and C-myc mRNA was dropped to each PC dot and reacted at 25 °C for 2 h. The mRNA probes hybridized with the target mRNA, and GO sheets were released form the mRNA probes. The detection device was washed with ultrapure water for three times to remove the GO sheets released form the mRNA probes. As a result, extraordinary luminescence recovery was obtained after adding target mRNA. The same detection results were obtained with the use of two kinds of experimental methods, and the second experimental method was chosen to conduct the following experiment. Briefly, mRNA probes were immobilized on PC dots first, and then each PC dot was added 1 μL of GO (0.3 mg mL 1 ) to bind with mRNA probes.
Consequently, a flexible device for the detection of mRNA was constructed. The detection of two kinds of mRNAs with the above device was conducted by adding target mRNA to the mRNA detection device. A 980 nm CW laser and an 808 nm CW laser were used to irradiate S7 the detection device and the luminescence images were captured by an unmodified camera phone.
Cell culture. MCF-7 cell was cultured in RPMI-1640 medium with 0.01 mg mL -1 bovine insulin. The cell lines were supplemented with 10% fetel bovine serum and 100 U mL -1 antibiotics penicillin/streptomycin and maintained at 37 °C in a 100% humidified atmosphere containing 5% CO 2 at 37 °C.
mRNA extraction from MCF-7 cell or tissues. mRNAs were extracted using EasyPure ® RNA Kit according to the manufacture's protocol.
(1) Sample pretreatment: For cell, 10 5 MCF-7 cells were disrupted firstly by add 300-600 μL of Lysis/Binding Solution to cell plate and then the sample was centrifuged at 12,000 × g for 5 min and the supernatant of the sample was removed to the RNase-free tube. For tissue, 10 mg of tissue were frozen with liquid nitrogen at first, and then were grinded into powder. After that, the powder was removed to an RNase-free tube and 1 mL of Binding Buffer 4 and 15 μL of Proteinase K were added. After the blending processing, the mixture reacted at 56 °C for 15 min. The sample was centrifuged at 12,000 × g for 5 min and the supernatant of the sample was removed to the RNase-free tube.
(2) Extraction of mRNA: 200 μL of 70% ethanol was added and mixed thoroughly. The sample was centrifuged at 12,000 × g for 30 sec and the supernatant was discarded. 500 μL of Cleaning Buffer 4 was added to the spin column and the sample was centrifuged at 12,000 × g for 30 sec and the supernatant was discarded. 500 μL of Washing Buffer 4 was added and the sample was centrifuged at 12,000 × g for 30 sec and the supernatant was discarded. Then, the spin column was centrifuged at 12,000 × g for 2 min to remove ethanol completely. The spin column was put to a new RNase-free tube and 100 μL of RNase-free water was added. The spin column was centrifuged at 12,000 × g for 2 min to elute mRNA to the tube.
Fresh samples from human breast cancer tissues and the corresponding non-tumor normal tissues were provided by Zhongnan Hospital (Wuhan University, China).

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Detection of mRNA extraction from MCF-7 cell or tissues. 2 μL of mRNAs extracted from MCF-7 cells or tissues were added to the mRNA detection device and reacted at 25 °C for 2 h.
Then, the detection device was washed with ultrapure water for three times to remove the GO sheets released form the mRNA probes. Finally, a 980 nm CW laser and an 808 nm CW laser were used to irradiate the detection device and the luminescence images were captured by an unmodified camera phone.
Luminescence image capture. A 980 nm CW laser and an 808 nm CW laser were used to irradiate the detection device. A mobile phone was used to capture the readout under different lighting conditions, using the camera's default autofocus and autoexposure settings.

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Supplementary Figures   Fig. S1. SEM image of PC dot.