Probe for detecting trace uranyl ions and portable ECL detector based on the same

Abstract

A high-sensitivity, high-selectivity and portable detection method for trace uranyl ion is described. The method has an ultralow detection limit of 11 pM/2.6 ppt and is useful in precise monitoring of the uranium content in agricultural and sideline products, foods, environments and so on. The test instrument is miniaturized and low in cost to achieve high-precision portable measurement in the field. A conjugated polymer with aggregation-induced emission (AIE) activity is synthesized, and prepared into Pdots, and a uranyl-responsive electrochemiluminescence (ECL) probe is developed by modifying the Pdots with DNA or RNA, which serves as an adsorption ligand of uranyl ion. The probe exhibits good biocompatibility. The ECL technology can be used in uranyl ion detection and the method has extremely high sensitivity. A uranyl ion probe with AIE activity is also disclosed, which can be applied in portable precise monitoring of trace uranyl ion by means of the ECL technology.

Claims

1. A probe for detecting trace uranyl ions, comprising conjugated polymer dots with AIE activity and a nucleotide sequence modified on the surface of the conjugated polymer dots, wherein the nucleotide sequence is a DNA and/or RNA sequence, and the conjugated polymer in the conjugated polymer dots has the structural Formula (1): ##STR00003## where R.sup.1 is an alkyl group, alkoxy group, substituted alkyl group or substituted alkoxy group containing 1-20 carbon atoms, in which the substituents in the substituted alkyl group and the substituted alkoxy group are independently selected from the group consisting of amino, carboxyl, hydroxyl, an ether bond, halo, a phosphoric acid group, an aldehyde group, a sulfonic acid group, a thioether bond, an ester group, amido, a Schiff base, an oximido, aryl, a sulfone group and a sulfoxide group; and n=2-300.

2. A method for preparing a probe for detecting trace uranyl ions according to claim 1, comprising steps of: (1) subjecting a first AIE active monomer of Formula (2) and a second AIE active monomer of Formula (3) to Suzuki coupling polymerization reaction to obtain a conjugated polymer of Formula (1), in which Formulas (2) and (3) are shown below: ##STR00004## where R.sup.1 is an alkyl group, alkoxy group, substituted alkyl group or substituted alkoxy group containing 1-20 carbon atoms, in which the substituents in the substituted alkyl group and the substituted alkoxy group are independently selected from the group consisting of amino, carboxyl, hydroxyl, an ether bond, halo, a phosphoric acid group, an aldehyde group, a sulfonic acid group, a thioether bond, an ester group, amido, a Schiff base, an oximido, aryl, a sulfone group and a sulfoxide group; R.sup.2 is a pinacol borate ester group, a trialkyltin group, a boric acid group, halo, ethynyl or vinyl; and R.sup.3 is halo, a pinacol borate ester group, a trialkyltin group, a boric acid group, ethynyl or vinyl; (2) injecting a solution of the conjugated polymer in an alcohol solvent into water, and removing the alcohol solvent, to obtain an aqueous solution of conjugated polymer dots; and (3) mixing the aqueous solution of the conjugated polymer dots with polyethylene glycol and a buffer; then adding a nucleotide sequence modified with an amino group at the 5 end and a cross-linking agent, where the nucleotide sequence is a DNA and/or RNA sequence; and mixing well and reacting to obtain the probe for detecting trace uranyl ions.

3. The preparation method according to claim 2, wherein in Step (2), the particle size of the conjugated polymer dots is 10 nm-200 nm.

4. The preparation method according to claim 2, wherein in Step (2), the concentration of the aqueous solution of the conjugated polymer dots is 0.01-1.00 mg/mL.

5. An ECL detector for detecting trace uranyl ions, wherein the ECL detector detects trace uranyl ions in the presence of an amine co-reactant reagent, and the ECL detector comprises a working electrode modified with the probe for detecting trace uranyl ions according to claim 1.

6. The ECL detector according to claim 5, comprising: an electric signal applying unit; a luminescence cell for accommodating a test solution and the amine co-reactant reagent, a photomultiplier tube directly facing the luminescence cell; and a signal amplifying and processing unit; wherein a positive and a negative electrode of the electric signal applying unit are respectively connected to the working electrode and a counter electrode which are positioned in the luminescence cell, and the photomultiplier tube is electrically connected to the signal amplification and processing unit.

7. The ECL detector according to claim 5, wherein the detection limit of uranyl ions is 11 pM.

8. The ECL detector according to claim 5, wherein the concentration of the amine co-reactant reagent is 5-100 mM.

9. The ECL detector according to claim 6, wherein the voltage applied by the electric signal applying unit is 0.50 V-2.00V.

10. A method for detecting trace uranyl ions, by using the ECL detector for detecting trace uranyl ions according to claim 5, comprising steps of: (1) detecting ECL signal intensities of uranyl ion standard solutions by the ECL detector, and establishing a correlation diagram between the uranyl ion concentrations and the ECL signal intensities according to the detection results; and (2) detecting an ECL signal intensity A.sub.x of a test solution by the ECL detector, where the content of uranyl ions in the test solution is unknown, and determining the uranyl ion concentration in the test solution according to the corresponding relationship of the ECL signal intensity A.sub.x in the correlation diagram.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram showing the synthesis route of a conjugated polymer with AIE activity of the present invention;

(2) FIG. 2 shows the test results by UV absorption spectrometry and fluorescence spectrometry of a THF solution of a conjugated polymer;

(3) FIG. 3 is a cyclic voltammogram of a conjugated polymer;

(4) FIG. 4 shows the test results by AFM and DLS of Pdots;

(5) FIG. 5 is a schematic diagram an ECL device;

(6) FIG. 6 shows the ECL signal test results and ECL spectrum of Pdots;

(7) FIG. 7 shows the zeta potential of Pdots before (A) and after (B) modification with the single-strand DNA;

(8) FIG. 8 is a schematic diagram of an ECL detector;

(9) FIG. 9 shows the ECL signal of Pdots in the presence of different concentrations of uranyl (UO.sub.2.sup.2+) and a calibration curve of the ECL intensity against the logarithmic concentration of uranyl;

(10) FIG. 10 demonstrates the RET process from uranyl to Pdots; and

(11) FIG. 11 illustrates the ECL signal intensity of interfering ions and uranyl.

LIST OF REFERENCE NUMERALS

(12) 1: working electrode, 2: counter electrode, 3: constant voltage DC power supply, 4: wire, 5: luminescence cell, 6: photomultiplier tube, 7: electrical signal applying unit, 8: signal amplification and processing unit.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(13) The specific embodiments of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are intended to illustrate the present invention, instead of limiting the scope of the present invention.

Example 1

(14) This example provides a method for preparing an ATE-active conjugated polymer. The synthesis route was shown in FIG. 1. The specific steps were carried out as follows:

(15) The synthesis of Compound 4 was as described in ACS Appl. Mater. Interfaces 2017, 9, 11546-11556. The synthesis of Monomer M-2 was as described in Polym. Chem. 2015, 6, 5070-5076.

(16) Synthesis of Monomer M-1: Compound 4 (2.28 g, 3 mmol), Compound 5 (3.08 g, 12 mmol), potassium acetate (2.40 g, 12 mmol), and Pd(dppf).sub.2Cl.sub.2 (0.3 g, 5% mmol) were dissolved in DMF (60 mL) and, heated to 120 C. under Ar atmosphere for 24 h. The reaction solution was poured into water (200 mL), and extracted with ethyl acetate. The organic phase was washed twice with water, and purified by column chromatography on silica gel (mobile phase, ethyl acetate:petroleum ether=1:30) to obtain the product (1.61 g, yield 64.0%). The NMR test results are shown below:

(17) .sup.1H NMR (400 MHz, CDCl.sub.3) 7.52 (d, J=8.1 Hz, 4H), 7.00 (d, J=8.1 Hz, 4H), 6.90 (d, J=8.7 Hz, 4H), 6.60 (d, J=8.8 Hz, 4H), 3.86 (t, J=6.6 Hz, 4H), 1.81-1.66 (m, 4H), 1.50-1.37 (m, 5H), 1.37-1.24 (m, 40H), 0.88 (t, J=6.8 Hz, 6H). .sup.13C NMR (100 MHz, CDCl.sub.3) 157.80, 147.35, 141.33, 138.76, 135.96, 134.10, 132.61, 130.84, 113.55, 83.66, 67.78, 31.82, 29.40, 29.33, 29.24, 26.09, 24.90, 22.67, 14.11.

(18) Synthesis of conjugated polymer: M-1 (0.16 g, 0.19 mmol), M-2 (0.10 g, 0.19 mmol), Pd(PPh.sub.3).sub.4 (0.04 g, 5% e.q.), and K.sub.2CO.sub.3 (1.3 g) were dissolved in toluene (15 mL), ethanol (8 mL) and water (4 mL). The mixture was further refluxed for 3 d. The organic phase was dried over Na.sub.2SO.sub.4. After the solvent being removed by a rotary dryer, the mixture was dissolved in a small amount of THF to be dropped into n-hexane (100 mL). The solvent was filtered to obtain the conjugated polymer (0.10 g, yield 53.8%). The .sup.1H NMR and GPC test results are shown below:

(19) .sup.1H NMR (400 MHz, CDCl.sub.3) 8.06-7.99 (m, 3H), 7.73-7.63 (m, 5H), 7.50-7.38 (m, 8H), 7.12-7.11 (m, 2H), 6.98-6.97 (m, 2H), 6.77-6.76 (m, 3H), 6.66-6.64 (m, 3H), 6.53-6.51 (m, 3H), 3.89-3.86 (m, 4H), 2.88 (s, 6H), 1.77-1.72 (m, 6H), 1.42-1.41 (m, 3H), 1.29-1.24 (m, 15H), 0.87-0.86 (m, 6H). GPC data: M.sub.w=19060, M.sub.n=16720, PDI=1.14.

(20) The photoelectric performance test results of the conjugated polymer prepared above are shown in FIG. 2. FIGS. 2A and 2B are the UV absorption spectrum of the conjugated polymer and the fluorescence spectra in THF-water mixtures with different water fractions. In FIG. 2B, the excitation wavelength is 370 nm, and the concentration of conjugated polymer in each solution is 110.sup.5 mol/L. As shown in FIG. 2A, the conjugated polymer exhibits an absorption peak of the conjugated backbone at 363 nm, and a shoulder peak at 421 nm, which could be attributed to the intramolecular charge transfer (ICT) caused by the electron donor-acceptor (D-A) structure. As shown in FIG. 2B, the conjugated polymer shows obvious AIE behavior in the deep-red/near-infrared region under an excitation wavelength of 370 nm. FIG. 3 shows a cyclic voltammogram (CV) data of the conjugated polymer in 0.1 M Bu.sub.4NBF.sub.4 CH.sub.2Cl.sub.2 solution tested by an GCE electrode, in which the two oxidation peaks at +0.487 V and +0.815 V can be attributed to the oxidation peaks of the TPE moiety. The reduction peak appears at 1.711 V, and the LUMO, HOMO, and band gap values can be calculated as 2.849 eV, 4.824 eV and 1.975 eV, respectively. The optical band gap from the emission spectrum was calculated as 1.890 eV (E.sub.g=1239.8/ (eV)).

Example 2

(21) The present invention provides a method for preparing the probe for detecting trace uranyl ions, which includes the following steps:

(22) The conjugated polymer prepared in Example 1 was dissolved in ethanol with a concentration of 0.15 mg/mL. Then the conjugated polymer solution (1 mL) was poured into water (10 mL), and the ethanol was removed by vacuum distillation to obtain an aqueous Pdots solution (10 mL).

(23) A PEG aqueous solution (5% w/v), and HEPES buffer (1 M) were added to the aqueous Pdots solution (0.15 mg/mL), and then NH.sub.2-DNA (1 M) and EDC (5 mg/mL) were added to the mixture and shaken for 0.5 h to obtain DNA-Pdots. The DNA sequence is: 3-taa ttc tgt gta tgt gtc tgt-5-NH.sub.2.

(24) In this example, the base sequences in the DNA sequence can be selected randomly, and the DNA sequence can also be replaced by an RNA sequence.

(25) FIG. 4(A) is an AFM image of Pdots; FIG. 4(B) shows the relative height of the black line in the AFM image; and FIG. 4(C) shows the DLS test result of Pdots. Both AFM image and DLS data show that the average particle size of Pdots is about 10 nm.

(26) Pdots were modified on a working electrode and assembled into a simple ECL device. As shown in FIG. 5, the modified working electrode was inserted into the luminescence cell, and 25 mM tri-n-propylamine (TPrA) buffer or 0.1 M PBS buffer (pH=7.4) was injected. A counter electrode and a reference electrode were inserted, and then the DNA-Pdots modified electrode was used as the working electrode. Test conditions: sweep speed: 100 mV/s, PMT: 600 V, .sub.ex=370 nm. When the potential was swept to +0.880 V, an optical signal was emitted, and when the potential was swept to +1.264 V, the optical signal was the strongest.

(27) As shown in FIG. 6A, Pdots cannot give obvious ECL signal in annihilation state. The obvious anodic ECL signal can be observed only when TPrA is added as a co-reactant reagent. The peak value of the ECL signal is +1.264V and the onset potential is +0.880V. The ECL mechanism is as follows:
TPrAe.sup..fwdarw.TPrAH.sup.+(1)
TPrAH.sup.+H.sup.+TPrA.sup.(2)
Pdotse.sup..fwdarw.Pdots.sup.+(3)
Pdots.sup.++TPrA.sup..fwdarw.Pdots*+Pr.sub.2N.sup.+HCCH.sub.2CH.sub.3(4)
Pdots*.fwdarw.Pdots+hv(5)

(28) As shown in FIG. 6B, the ECL spectrum of Pdots is basically overlapped with its fluorescence spectrum, which indicates that the ECL mechanism follows the band gap emission model.

(29) Single-stranded DNA is modified on Pdots for using the phosphate group on the DNA strand as a ligand for binding uranyl ions. The ECL signal of Pdots can be enhanced through the resonance energy transfer (RET) process from uranyl ions to Pdots, thereby realizing the high sensitive and selective detection of uranyl ions. FIG. 7 shows the zeta potential of Pdots before (A) and after (B) modification with single-stranded DNA. The zeta potential of Pdots is 22.1 mV before modification with DNA. After modification with DNA, the potential rises to 37.5 mV, indicating the successful modification of Pdots.

(30) As shown in FIG. 8, this example also provides an ECL detector for detecting trace uranyl ions. The ECL detector comprises an electrical signal applying unit, a luminescence cell, a photomultiplier tube, and a signal amplifying and processing unit. A positive and a negative electrode of the electric signal applying unit are respectively connected to the working electrode and the counter electrode, where the working electrode is modified with the above-mentioned probe for detecting trace uranyl ions. The luminescence cell is used for accommodating a test solution and an amine co-reactant reagent, the working electrode and the counter electrode are positioned in the luminescence cell, the photomultiplier tube is arranged facing directly the luminescence cell, and the photomultiplier tube is electrically connected to the signal amplification and processing unit.

(31) The working electrode is modified as follows:

(32) GCE electrodes were polished with Al.sub.2O.sub.3 powder. The DNA-Pdots obtained in Example 2 were mixed with uranyl solutions (different concentrations), natural water samples or interfering ion solutions. The GCE electrodes were immersed in the above mixed solution to allow the DNA-Pdots to be modified on the GCE electrode.

(33) FIG. 9 shows the ECL signals of Pdots in the presence of different concentrations of UO.sub.2.sup.2+ (A), and the calibration curve of the ECL intensity against the logarithmic concentration of uranyl ion (B). Sweep speed: 100 mV/s, PMT: 600 V. As shown in FIG. 9A, as the uranyl ion concentration increases from 0.05 nM to 100 nM, the ECL signal intensity of Pdots gradually increased. The ECL signal intensity I is obviously linear with the logarithmic concentration C of uranyl in this region (FIG. 9B, I=8109.7+4108.31 gC, R.sup.2=0.996). The detection limit could be calculated as 11 pM/2.6 ppt, which is at least two orders of magnitude lower than that of the currently known uranyl ion luminescent probe, especially the known AIE-active probe.

(34) In order to confirm the RET process from uranyl ions to Pdots, FIG. 10A shows the ECL signal of uranyl ion in 0.1 M PBS (pH 7.4) containing 25 mM TPrA, and FIG. 10(B) shows the UV-vis spectrum (a) of the conjugated polymer solution in THF and the ECL spectrum (b) of uranyl ions in 0.1 M PBS (pH 7.4) containing 25 mM TPrA. Sweep speed: 100 mV/s, PMT: 600 V. As shown in FIG. 10A, with 25 mM TPrA as a co-reactant regent, the uranyl ion shows a significant ECL signal in the anodic region, with an emission peak at +1.120 V with the onset potential as +0.791 V. The ECL spectrum of UO.sub.2.sup.2+ ion gives an emission peak around 500 nm, which overlaps with the UV-vis absorption spectrum of the conjugated polymer in the region of 400 to 560 nm. This phenomenon indicated the RET process from uranyl ion to Pdots (FIG. 10B).

(35) To confirm the selectivity of the probe, several interfering ions were selected for comparison with UO.sub.2.sup.2+ ions. K.sup.+, Ca.sup.2+, Na.sup.+, Mg.sup.2+ and Sr.sup.2+ are main cations contained in sea water. Eu.sup.3+ is an ion with ECL signal, which is closely related to the adsorption and detection of UO.sub.2.sup.2+ ions. Cs.sup.+ often coexists with UO.sub.2.sup.2+, and Cu.sup.2+, Fe.sup.3+ and Pb.sup.2+ are representative of heavy metals in the environment. Compared with UO.sub.2.sup.2+ ions with a concentration of 0.5 nM or 5 nM, these interfering ions have almost no significant signal enhancement even in a solution of 50 nM (FIG. 11). The selectivity of this probe can be attributed to i) the specific adsorption of UO.sub.2.sup.2+ ions by the phosphate groups on the DNA strand; and ii) the non-ECL property of interfering ions in anodic region. The experimental results confirm the excellent selectivity of ECL probe for UO.sub.2.sup.2+ based on Pdots.

(36) In order to further confirm the applicability of this probe, the concentration of uranyl ions in some practical water samples was determined in this example, including water samples from Bohai Sea (Tianjin, China), Luoma Lake (Xuzhou, China), Dushu Lake (Suzhou, China), and Qiandao Lake (Hangzhou, China) (Table 1). It can be observed that the results obtained by ECL is quite close to ICP-MS, indicating the practical application of this new ECL probe in environment field.

(37) TABLE-US-00001 TABLE 1 Comparison of the uranyl concentration values obtained by ECL and ICP-MS Test result by ICP-MS (ppb) Test result by Result RSDs Water source ECL (ppb) (ppb) (%) Sea Bohai Sea (Tianjin) 3.24 0.05 2.05 1.92 water Fresh Luoma Lake (Xuzhou, 1.48 0.02 1.25 0.98 water Jiangsu Province) Dushu Lake (Suzhou, 0.38 0.08 0.50 0.32 Jiangsu Province) Qiandao Lake (Hangzhou, 0.21 0.01 0.15 0.35 Zhejiang Province)

(38) While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. It should be appreciated that some improvements and variations can be made by those skilled in the art without departing from the technical principles of the present invention, which are also contemplated to be within the scope of the present invention.