High-throughput imaging assay of multiple proteins via target-induced DNA assembly and cleavage† †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c4sc03809f Click here for additional data file.

A versatile imaging strategy integrated with target-induced DNA assembly and cleavage was designed for an assay for multiple proteins.


Materials and reagents
The oligonucleotides were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd.

Apparatus
Cooled low-light CCD with high resolution (BioImaging Systems Chemi HR 410 camera, UVP, USA) was used for CL imaging assay (CLIA). IFFM-E luminescent analyzer (Remax, China) was used to study the kinetic behavior of the CL reaction. Luxscan-10k/A microarray scanner (Capitalbio, China) was used for FL imaging assay (FIA).

Fabrication of disposable DNA chip
Briefly, a predesigned hydrophobic photo-inactive film with 96 holes (1.5 mm diameter, 6 rows × 16 columns with edge-to-edge separation of 2 mm) was firstly fixed on an aldehyde-modified glass slide, and 1 μL of 5 nM DNA1-FITC was dropped on the formed cell to incubate overnight at 4 °C. The 96 cells were then thoroughly washed with washing buffer, and the unreacted aldehyde groups were S3 blocked with blocking buffer for 2 h. After washing, the disposable DNA chip was obtained and stored in 0.01 M pH 7.4 PBS3 at 4 °C.

Preparation of affinity probes
The DNA-labeled affinity probes were prepared with a modified coupling procedure. 1 Antibody (2 mg mL -1 ) was firstly activated with a 20-fold molar excess of SMCC in PBS1 for 2 h at room temperature, and purified by ultrafiltration using a 100 KD millipore (10000 r, 10 min). In parallel, 12 μL of 100 μM thiolated DNA3 and DNA4 were reduced with 16 μL of 100 mM DTT in PBS1 at 37 °C for 1 h, and purified by ultrafiltration using a 10 KD millipore (10000 r, 10 min). The reduced DNA3 was then mixed with DNA2 to hybridize for 1 h at 25 °C. After the activated antibody was incubated with DNA4 or DNA3/DNA2 duplex in PBS2 overnight at 4 °C, the excess DNA4 or unreacted DNA3/DNA2 duplex was removed by ultrafiltration using a 100 KD millipore (10000 r, 10 min) for several times to obtain the DNA-labeled affinity probes, which were collected at a concentration of 6.0 µM in PBS2 and diluted with PBS2 for 40 folds prior to use.

PAGE and mass spectroscopic analysis of affinity probes
For polyacrylamide gel electrophoresis (PAGE) analysis, a 10% native polyacrylamide gel was prepared using 5×TBE buffer. The loading sample was the mixture of 7 μL of obtained DNA or affinity probe, 1.5 μL of 6×loading buffer, and 1.5 μL of UltraPowerTM dye. Before injection into the polyacrylamide hydrogel, the loading sample was placed for 3 min. The gel electrophoresis was run at 90 V for 1 h. The resulting board was illuminated with UV light and photographed with a Molecular Imager Gel Doc XR.

FIA of proteins
To obtain the calibration curves for FIA of AFP, CA 125, CA 199 and CEA, 1.0 μL of different standard antigen solutions at known concentration were firstly mixed with 29 μL of incubation solution containing 5.0 nM corresponding DNA3/DNA2 duplex-linked antibody 1 (Ab-DNA3/2), 5.0 nM corresponding DNA4-linked antibody 2 (Ab-DNA4) and 2 U Nt.BbvCI, respectively. 1 μL of the as-prepared mixtures were immediately dropped into the cells on DNA chip and incubated for 30 min.
After washing, the image for recording the fluorescent signals of FITC in the immobilized DNA1-FITC was recorded at an excitation wavelength of 532 nm. The fluorescent signal at each spot was S4 read with a VisionWorksLS image acquisition and analysis software (UVP) after adjusting the saturation of the image, and calculated as the mean pixel intensity within a square around the spot center.

CLIA of proteins
CLIA was carried out with a similar procedure described above. After the DNA chip was incubated with the mixtures of standard solution or sample, 5.0 nM Ab-DNA3/2, 5.0 nM Ab-DNA4, 5.0 nM HRP-Ab FITC and 2 U Nt.BbvCI for 30 min and washed, 1 μL of CL substrate was delivered into each cell to trigger the CL reaction. The CL image was recorded by a CCD with six 30-s exposure times for dynamic integration of 3 min. The CL signals were automatically identified with UVP and calculated as the mean pixel intensity in the squares around the spot centers.