Supporting information

Figure S1. (A) SEM image of MMPs. The horizontal scale bar is 5 μm. (B) Histogram of the size distribution of

MMPs.

Figure S2. Bright-field Image of MMPs. The number of diluted MMPs was 363. The horizontal scale bar is 50 μm.

Figure S3. Comparison of hydrodynamic size (A) and zeta potential (B) of MMPs before and after modification

with Ab/β-gal.

Figure S4. (A) Bright-field images of (a) MMPs/Ab (c) MMPs-Ab/β-gal reacting with T RITC-conjugated Donkey

Anti-Mouse IgG. Fluorescence images of (b) MMPs/Ab (d) MMPs-Ab/β-gal reacting with T RITC-conjugated

Donkey Anti-Mouse IgG. The horizontal scale bar is 50 μm. (B) Fluorescence intensity of FDG reacted with (1)

MMPs/β-gal (2) MMPs-Ab/β-gal. Inset: A Fluorescence image of the FDG reacted with (1) MMPs/β-gal (2) MMPs-

Ab/β-gal.

Figure S5. (A) Fluorescence spectra of various concentration of β-gal reacting with FDG. Different concentrations

of β-gal were reacted with FDG at 37 °C for 10 min. (B) The calibration curves of fluorescence intensities versus

β-gal concentrations. (C) Fluorescence spectra of various concentration of TRITC-conjugated Donkey Anti-Mouse

IgG. (D) The calibration curves of fluorescence intensities versus TRITC-conjugated Donkey Anti-Mouse IgG

concentrations.

Figure S6. Fluorescence images of (A) unmodified and (B) modified microwell of microfluidic array chip after the

reaction with TRITC-conjugated Donkey Anti-Mouse IgG. Horizontal scale bar is 25 μm. (C) Fluorescence spectra of

the initial and collected solution.

Figure S7. Evaluation of the evaporation loss when the outlet and inlet are not blocked by hot glue. (A) Fluorescence images of fluorescein sequestered in microwells for 15, 30, 45, and 60 min. Horizontal scale bar is 200 μm. (B)Volume change of fluorescein in microwells with an average volume loss of 58.77%.

Figure S8. (A) Fluorescence images of the microwells after the introduction and sequestration of diluted modified

MMPs and FDG into microwells for (a) 15, (b) 30, (c) 45 and (d) 60 min. The horizontal scale bar is 200 μm. (D)

Scatter diagram of mean fluorescence intensity of three selected microwells.

Figure S9. Fluorescence images of the microfluidic array chip in contrast analysis (A) without being rinsed and (B)

with being rinsed. The horizontal scale bar is 1000 μm. (C) Optimization of the flow velocity of the washing buffer.

The digital detection of 0 (black) and 10 fg/mL (red) AFP with different flow velocity: 50, 100, 200, 400, 800 μL/h,

respectively. The optimized flow velocity is 100 μL/h.

Figure S10. Mean fluorescence intensity distribution of 900 microwells with concentration 0, 1 fg/mL, 10 fg/mL,

100 fg/mL.

Table S1. Comparing the Poisson noise-associated coefficient of variation (CV = √n/n, n is the average number of

AFP) with the measurement CV over three measurements.

Concentration of [AFP] (fg/mL) Measurement CV Poisson noise CV

1 0.1619 0.84625 0.0857 0.3784

10 0.0271 0.267625 0.0832 0.169350 0.0517 0.119775 0.0339 0.0977

100 0.0177 0.0846