*2.3. Synthesis*

#### 2.3.1. Synthesis of 3-(3-Bromopropoxy)-4-methylthiophene (**1**)

The monomer **1** was synthesized according the procedure described in the previous work reported by our group [18]. Yield 81%; 1H NMR (400 MHz, CDCl3) δ 6.72 (s, 1H), 6.08 (s, 1H), 3.96 (m, 2H), 3.48 (m, 2H), 2.23 (m, 2H), 1.99 (s, 3H).

#### 2.3.2. Synthesis of 3-(4-Methyl-3-thienyloxy)propyltrimethylammonium Bromide (**2**)

Trimethylamine (10 mL) was added into a solution of **1** (2.68 g, 11.4 mmol) in THF (100 mL). The mixture was stirred 48 h at room temperature, and then evaporated to dryness. The crude product was washed with THF to give **2** (2.62 g, 78% yield). 1H NMR (400 MHz, *d*6-DMSO) δ 7.01 (s, 1H), 6.48 (s, 1H), 4.15 (m, 2H), 3.51 (m, 2H), 3.14 (s, 9H), 2.30 (m, 2H), 2.06 (s, 3H).

#### 2.3.3. Synthesis of Copolymer (**3**)

Copolymer **3** was synthesized according the procedure as following: anhydrous FeCl3 (649 mg, 4 mmol) was added to the freshly dry CHCl3 (20 mL), and the mixture was stirred for 30 min at room temperature under N2 atmosphere. Then, the solution of **1** (235 mg, 1 mmol) and **2** (294 mg, 1 mmol) dissolved in dry CHCl3 (10 mL) was added dropwise to the above as-prepared FeCl3 solution. The mixture was stirred for 48 h at 35 ◦C. The reaction mixture was then concentrated to about 2 mL. The residue was precipitated by addition of MeOH (200 mL). The precipitate was collected by filtration, and the resulting crude product was extracted by Soxhlet extraction with MeOH for 24 h to remove possibly residual FeCl3. The residual solid was filtrated and dried under reduced pressure to give copolymer **3** (331.6 mg, 63%). GPC (H2O, pullulan standard): *M*n: 6.3 kDa, PDI: 1.819.

#### 2.3.4. Synthesis of Copolymer **L**

Copolymer **3** (200 mg) was dissolved in a mixed solvent of DMF (30 mL) and THF (20 mL). 3-Pyridineboronic acid (300 mg, 2.44 mmol) was then added into the above solution. The mixture was stirred at 70 ◦C under a N2 atmosphere for 48 h. The mixed solution was concentrated to about 2 mL, and the residue was then dropwise added into THF (60 mL). The resultant precipitate was collected by filtration, washed with THF (50 mL × 3), and dried under vacuum at room temperature to obtain polymer **L** (211 mg, 86% yield) as a dark-red solid.

#### *2.4. Sample Preparation for Spectroscopic Analysis*

All spectroscopic experiments were carried out at 25 ◦C. The stock solution of the probe (**L** = 2.0 × 10−<sup>3</sup> M) in water was diluted to 50 μM for UV-Vis absorption spectrum and 10 μM for photoluminescence spectrum.

#### *2.5. Cell Culture and Cytotoxicity Assays*

HeLa cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 ◦C in a humidified 5% CO2–95% air atmosphere.

The cytotoxicity of **L** was evaluated by typical MTT assay. HeLa cells (1 × 10<sup>4</sup> cells/well) were seeded onto 96-well plates in 100 μL DMEM and allowed to attach for 24 h. After cell attachment, the medium was removed and the cells were washed with PBS three times, then 100 μL of fresh medium containing **L** with different concentrations ranging from 0 to 50 μM was added and incubated for 24 h. After 24 h incubation, the cell viability was evaluated by MTT assay.

## *2.6. Fluorescence Imaging*

HeLa cells (1 × 10<sup>5</sup> cells/well) were seeded onto petri dish in 2 mL DMEM and allowed to attach for 24 h. After cell attachment, the medium was replaced with 2 mL of fresh medium containing **L** (10 μM, 2 mL) for 2 h, then the medium was removed and the cells were washed with PBS three times. Fluorescence images were recorded with a confocal laser scanning microscope (CLSM) (Olympus FV1000-IX81, Tokyo, Japan). The excitation wavelength used was 405 nm, and all images were analyzed with Olympus FV1000-ASW.

#### **3. Results and Discussion**

#### *3.1. Synthesis of L*

The synthetic route for the probe **L** is outlined in Scheme 2. The traditional chemical oxidative polymerization with FeCl3 in chloroform in the presence of equivalent **1** and **2** gave the random copolymer product **3** with a moderate number-average molecular weight of 6.3 kDa, determined by GPC using pullulan as the standard and H2O as the eluent, respectively. Subsequently, 3-pyridineboronic acid groups were linked to the side chain of **3** by nucleophilic substitution reaction to produce the target **L**. The strong absorption peaks at 1,360 and 1,485 cm<sup>−</sup><sup>1</sup> in FTIR of **L** were assigned to the characteristic symmetric and unsymmetric stretching vibration of the B–O bond, respectively (Figure S1, Supplementary Materials), suggesting that the boronic acid groups were successfully introduced into the side chain of **L**. The ratio of the three repeated units in **L** was determined to be 0.17:0.83:1 for Br, boronic acid and quaternary ammonium contained ones, respectively, by inductively coupled plasma (ICP) analysis. In other words, the molar content of boronic acid-containing moieties in the total polymer was about 41.5%.

**Scheme 2.** Synthesis of the probe **L**. (**i**) N(CH3)3, THF, rt, 48 h; (**ii**) FeCl3, CHCl3, 35 ◦C, 48 h; (**iii**) 3-Pyridineboronic acid, DMF/THF (3:2 *v*/*v*), N2, 70 ◦C, 48 h.

#### *3.2. Screen of pH of Assay System*

Since pH values of the aqueous solution influences strongly the formation of ester between boronic acids and vicinal diols [6,26], the pH conditions for ATP sensing using **L** as a probe were first optimized. The pyridinium hydroxyboronate moiety in our probe **L** has a very low pKa value of about 4.0 [30]. Thus, the weakly alkaline conditions with pH values ranging from 7.0–10.0 were carefully assessed. As shown in Figure S2a (Supplementary Materials), the changes on absorption spectra of **L** (50 μM) in water were almost ignored within the total tested pH ranges. Upon addition of 1 equiv. of ATP to **L** aqueous solution (50 μM) with different pH values, the absorption spectra of solutions at pH = 7.4, 8.0, and 9.0 gave the significant responses (Figure S2b, Supplementary Materials). Based on these results, the aqueous solution of physiological pH 7.4 was chosen as assay system for the evaluation of sensing properties of **L**.
