Multiple Emitting Amphiphilic Conjugated Polythiophenes‐Coated CdTe QDs for Picogram Detection of Trinitrophenol Explosive and Application Using Chitosan Film and Paper‐Based Sensor Coupled with Smartphone

Abstract Novel multiple emitting amphiphilic conjugated polythiophene‐coated CdTe quantum dots for picogram level determination of the 2,4,6‐trinitrophenol (TNP) explosive are developed. Four biocompatible sensors, cationic polythiophene nanohybrids (CPTQDs), nonionic polythiophene nanohybrids (NPTQDs), anionic polythiophene nanohybrids (APTQDs), and thiophene copolymer nanohybrids (TCPQDs), are designed using an in situ polymerization method, which shows highly enhanced fluorescence intensity and quantum yield (up to 78%). All sensors are investigated for nitroexplosive detection to provide a remarkable fluorescence quenching for TNP and the quenching efficiency reached 96% in the case of TCPQDs. The fluorescence of the sensors are quenched by TNP through inner filter effect, electrostatic, π−π, and hydrogen bonding interactions. Under optimal conditions, the detection limits of CPTQDs, NPTQDs, APTQDs, and TCPQDs are 2.56, 7.23, 4.12, and 0.56 × 10−9 m, respectively, within 60 s. More importantly, portable, cost effective, and simple to use paper strips and chitosan film are successfully applied to visually detect as little as 2.29 pg of TNP. The possibility of utilizing a smartphone with a color‐scanning APP in the determination of TNP is also established. Moreover, the practical application of the developed sensors for TNP detection in tap and river water samples is described with satisfactory recoveries of 98.02−107.50%.


S4
added in one portion. The reaction mixture was refluxed for 8 h and water was collected azeotropically in the Dean-Stark trap. The mixture was allowed to cool to 40-45 °C, filtered to remove the boric acid present in the reaction mass and further cooled to 25-35 °C. After stirring for 1 h at 25-35 °C, toluene was decanted, and then the resulting crude material was dissolved in methanol (50 mL). Distillation afforded the product (1.01 g, yield 94.92%) as syrup. FT-IR (KBr, Figure S1

Synthesis of monomer 3
At 0 °C, chlorosulfonic acid (0.583 g, 5 mmol) was added to a solution of monomer 2 (2.124 g, 1 mmol) in dichloromethane (20 mL), and the resulting solution was stirred at room temperature overnight. Then, the solution was concentrated under vacuum, and ether was added to it. The resulting precipitate was filtered and washed with ether three times to get THP-PEG-OSO 3 H as a gummy solid. THP-PEG-OSO 3 H is treated with sodium hydroxide solution to convert the sulfonic acid into the sodium salt and afford monomer 3 (1.62 g, yield 73.50 %). FT-IR (KBr, Figure S3
The mixture was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was concentrated to 5 mL. The residue was poured into 200 mL of absolute diethyl ether under stirring and then filtered. The precipitate was washed with absolute diethyl ether and dried to give compound monomer 3 (0.39 g, yield 83.15%) as a yellow powder. FT-IR (KBr, Figure S7

Synthesis of CdTe QDs
The synthesis of CdTe QD was carried out according to the procedure described in the literature with small modifications [1] . Briefly, Te powder (0.1 mmol), NaBH 4 (0.2 mmol) and 0.5 mL of water were mixed and heated at 55 o C for 30 min until the black Te disappeared and the pink color of the NaHTe precursor was produced. At the same time CdCl 2 (1 mmol), MAA (2 mmol), and 100 mL of distilled water were mixed in a three-neck flask to form a cadmium precursor. The pH of the solution was adjusted to 10.5 using 1.0 N NaOH under vigorous stirring. The mixture was heated to 100 °C under stirring. Meanwhile, the NaHTe precursor solution was quickly injected into the cadmium precursor solution. The molar ratio of Cd 2+ : Te 2-: MAA was 1:0.1:2. The reaction medium was refluxed at 100 °C for 4 h and then cooled to room temperature. CdTe QDs were precipitated from the solution by added isopropanol at the volume ratio of 1:1 (isopropanol: water) followed by centrifugation at 4000 rpm.

Synthesis of CdTe QDs coated with cationic, nonionic, anionic polythiophenes and thiophene copolymer via in situ polymerization in aqueous solution (CPTQDs, NPTQDs,
APTQDs, and TCPQDs) S7 CPTQDs, NPTQDs and APTQDs were synthesized according to our previously reported procedures with some modification [2] . Briefly, each of monomers (1, 2 or 3) (0.03 mmol) was dissolved in 15 mL aqueous solution. The resulted solution was added to 5 mL of CdTe QDs (pH 7) and stirred for 30 min under a nitrogen atmosphere, then (NH 4 ) 2 S 2 O 8 (0.6 mmol) was added drop-wise into the mixture. The mixture was stirred for 24 h at 25 ± 1 °C. The same procedure was carried out for the synthesis of TCPQDs using monomer 1 (0.03 mmol),  nm was used to excite the samples to record their FL spectra. The QY was calculated using the equation (1) below [3] .
where x is the QY, I is the integrated fluorescence intensity, A is the absorbance, and n is the refractive index of the solvent; r denotes the standard and x denotes the sample.

Biocompatibility
To assess the biocompatibility of the CPTQDs, NPTQDs, APTQDs and TCPQDs in comparison with equivalent content of bare CdTe QDs, an MTT cell assay was performed on the HeLa cells. Briefly, HeLa cells were plated at a density of 1×10 4 cells per well in a 96well plate, and then incubated for 24 h at 37 °C under 5% CO 2 to allow the cells to attach to the wells. The CPTQDs, NPTQDs, APTQDs and TCPQDs were sterilized by autoclaving, and two different concentration (400 and 600 µg/mL) from QDs coated amphiphilic polymers were added to the culture wells to replace the original culture medium and were incubated for another 24 h in 5% CO 2 at 37 °C. For comparison the equivalent content of bare CdTe QDs (Table S3) were calculated based on TGA results (Figure 1d) and were added to the culture wells to replace the original culture medium and were incubated for another 24 h in 5% CO 2 at 37 °C. Next, 10 µL of MTT solution (5mg/mL) was added to each well (containing different amounts of the CPTQDs, NPTQDs, APTQDs and TCPQDs and their equivalent of CdTe QDs, followed by incubation for 4 h inside a CO 2 incubator at 37 °C. After incubation, the medium was removed, and the formed formazan crystals were dissolved in 100 µL of S9 DMSO/ethanol mixture (1:1). A Tecan Infinite M200 monochromator-based multifunction microplate reader was used to measure the OD 570 (Abs value) of each well with background subtraction at 540 nm. At least three independent experiments were performed in each case.
The following equation (2) was applied to calculate the viability of cell growth [4] :

Fluorescence sensing of TNP explosive
Stock solutions (0.01 mM) of CPTQDs, NPTQDs, APTQDs, and TCPQDs were prepared in PBS buffer (pH 7.0). Different concentrations of TNP were mixed with 1.5 mL of the respective sensor solutions such that the total volume amounted to 3 mL, and the samples were incubated for 60 s at room temperature before detection. The fluorescence spectra of the samples were measured with excitation at 340, 350, 360, and 380 nm, for the CPTQDs, NPTQDs, APTQDs, and TCPQDs, respectively. All the measurements were carried out in triplicate.

Determination of selectivity to TNP
This method entailed selecting several nitroaromatic explosives (Scheme S1) and metal ions as coexisting substances to investigate the selectivity of TNP. The concentration of each explosive was 50 µM, whereas the concentration of metal ions was selected as 1 mM.
The selected detection conditions were the same as those mentioned above.

TRPL studies
Excited state lifetime decay profiles of CPTQDs, NPTQDs, APTQDs and TCPQDs were obtained in both solvents before and after TNP addition via 375 nm pulse excitation and emission at 425, 430, 460, and 510 nm in aqueous medium, respectivly. The decay profiles were bi-exponentially fitted and for uniformity in results average lifetime were considered.

Determination of TNP in enviromental water samples
River and tap water samples were utilized to investigate the practical applicability of the developed method for the detection of TNP. Samples of river water were collected from the Changwon River (South Korea). Both types of water samples were filtered twice to remove any solid suspensions and then centrifuged at 4000 rpm for 15 min. The spiked river and tap water samples were diluted 50-fold with different concentrations of TNP (0.04, 0.80, and 1.60 µM) and added to 0.01 mM solutions of the CPTQD, NPTQD APTQD, and TCPQD sensors, respectively. The samples were incubated for 60 s at a pH of 7.0 before detection and S10 each experiment was repeated five times under the same conditions to determine the relative standard deviations (RSDs).

Preparation of the paper-based sensor
Whattman filter paper (70 mm diameter) was immerged in the TCPQDs (10 -4 M) solution for 60 min. The filter paper was then removed from the solution and dried at room temperature.
TCPQDs coated filter papers were then cut into desired number of pieces (1 cm × 1 cm) and a 10 L of TNP solution with various concentrations was dropped into the obtained filter paper strips, and the solvent on the filter paper strips was naturally evaporated at room temperature.
Paper strips were then visualized under 365 nm UV light. Furthermore, fluorescence color image was taken with a smartphone, and then RGB intensities of the image can be directly output by using a custom developed PAD Analysis APP, which can be readily downloaded to the smartphone online.

Preparation of fluorescence film-based sensor
In a 50 mL beaker containing TCPQDs (15 mg) and purified chitosan (300 mg), 30 mL Milli-Q water and 300 µL of acetic acid were added followed by continuous stirring for about 15 min to ensure complete dissolution of chitosan. This leads to the formation of highly viscous liquid that was spread on pre-cleaned glass plate/petri dish and dried at room temperature. A homogeneous transparent film was obtained that could be easily lifted using forceps for sensing purposes. For contact mode sensing, 5 mg of TNP was rubbed with the left hand thumb and brushed properly to remove all visible TNP particles. Left thumb was then pressed onto the film for 10 sec, kept aside and the impression observed under UV light.
Right hand thumb was used as control without using TNP. S11 Scheme S2. Schematic representation the detection of TNP using the CPTQDs, NPTQDs, and APTQDs sensor through IFE and molecular interactions mechanism. S12 Table S1. Characterization of different amphiphilic conjugated polythiophenes coated QDs a Measured in aqueous solution with a concentration of 100 µM in water. b Measured in aqueous solution with rhodamine B as standard.

Samples
Size, nm potential, mV

Reproducibility and stability of the sensors
The reproducibility of CPTQDs, NPTQDs, APTQDs and TCPQDs was investigated by the measurement of the response to 5 M from TNP. The relative standard deviations (RSDs) of the ten successive measurements were 1.98, 2.75, 2.04, and 1.62%, respectively, Figures. (S21a-d). In addition, the stability of the sensors was investigated after being stored at 4 °C for more than 120 days. The CPTQDs, NPTQDs, APTQDs and TCPQDs responses were stable and maintained 90, 92, 88% and 95% activity with a low deviation of 5, 4, 5 and 4%, respectively, indicating excellent stability of the sensors for the determination of TNP.