Gold/quantum dot nanoprobe for detecting active ricin in complex matrix and application thereof

Abstract

The present disclosure discloses a gold/quantum dot nanoprobe for detecting active ricin in a complex matrix and application thereof. The gold/quantum dot nanoprobe is a nanoprobe formed by utilizing gold nanoparticles and quantum dots, which are modified by single strand oligodeoxynucleotides (ssODN), to form double strand oligodeoxynucleotides in a base pairing hybridizing mode and assembling the gold nanoparticles and the quantum dots into a core-satellite structure. According to the present disclosure, the gold/quantum dot nanoprobe is used for detecting the active ricin, has a limit of detection of 7.46 ng/mL, is high in accuracy and good in reliability, and does not require large-scale equipment and complex operations. In order to further eliminate the false positive result, the present disclosure further provides a method for enriching ricin in a complex sample by utilizing magnetic beads. In a case that specific active ricin concentration does not need to be known, the gold/quantum dot nanoprobe provided by the present disclosure can implement naked eye visual detection by the quenched and switch-on operations of fluorescence.

Claims

1. A gold/quantum dot nanoprobe for detecting active ricin, comprising gold nanoparticles modified by a linker P1 containing single strand oligodeoxynucleotides (P1-AuNPs), quantum dots modified by a linker P2 containing single strand oligodeoxynucleotides (P2-QDs) and single strand oligodeoxynucleotides P3, wherein: the P1, the P2 and the P3 form double strand oligodeoxynucleotides by base pairing hybridization, the gold nanoparticles and the quantum dots are linked to be assembled into a core-satellite structure with the gold nanoparticles being a core and the quantum dots surrounding the gold nanoparticle core; a 5′ terminal of the linker P1 is sulfydryl and is linked with the surfaces of gold nanoparticles AuNPs to obtain the P1 modified gold nanoparticles (P1-AuNPs) and a 3′ terminal is a deoxynucleotide sequence L1; a 3′ terminal of the linker P2 is amino and is linked with quantum dots QDs to obtain the P2 modified quantum dots (P2-QDs) and a 5′ terminal is a deoxynucleotide sequence L2; the linkers P1 and P2 are respectively subjected to pairing hybridization with deoxynucleotide sequences L1′ and L2′ at both ends of the P3 to form a double strand structure to link the AuNPs and the QDs; a condensed structural formula of the linker P1 is 5′-SH-alkylene-poly(dT.sub.n1)-L1-3′, a condensed structural formula of the linker P2 is 5′-L2-poly(dT.sub.n2)-alkylene-NH.sub.2-3′, and a condensed structural formula of the linker P3 is 3′-L1′-poly(dA.sub.n3)-L2′-5′, wherein the number of carbon atoms of alkylene is an integer within a range of 4 to 12; poly(dT.sub.n1) and poly(dT.sub.n2) represent chains of deoxynucleotides of which a base is thymine (T), and n1 and n2 represent numbers of deoxynucleotides of which the base is the thymine (T) and are independently 6 to 15; poly(dA.sub.n3) represents a sequence of deoxynucleotides of which a base is adenine (A), n3 represents a number of deoxynucleotides of which the base is the adenine (A), and the number is an integer within a range of 9 to 21; L1 represents a sequence of 6 to 15 deoxynucleotides, wherein the number of the deoxynucleotides of which the base is the adenine (A) is at least 40% of a total number of the deoxynucleotides, and the number of the deoxynucleotides of which the base is the thymine (T) is at least 40% of the total number of the deoxynucleotides; L2 represents a sequence of 9 to 18 deoxynucleotides; in the sequence L1 at one end of the linker P1, the deoxynucleotides of which the base is the adenine (A) and the deoxynucleotides of which the base is the thymine (T) are alternately arranged to form a sequence of . . . ATATATAT . . . ; and the sequence L1′ at one end of the linker P3 is subjected to pairing hybridization with the sequence L1 of the P1 and is subjected to pairing hybridization with one part of the sequence L2 of the P2.

2. The gold/quantum dot nanoprobe according to claim 1, wherein the linkers P1 and P2 independently contain 18 to 30 deoxynucleotide units, the P3 contains 33 to 42 deoxynucleotide units, and a sequence of 12 to 15 deoxynucleotides of which a base is adenine (A) is positioned in the middle of the P3.

3. The gold/quantum dot nanoprobe according to claim 1, wherein a carbon atom number of alkylene is an integer within a range of 6 to 9, n1 and n2 are integers within a range of 9 to 12, n3 is an integer within a range of 12 to 15, L1 represents a sequence of 9 to 12 deoxynucleotides, and L2 represents a sequence of 12 to 15 deoxynucleotides.

4. The gold/quantum dot nanoprobe according to claim 1, wherein the linkers P1, P2 and P3 have structures in the following table and P1 is P1(a), P1(b), or P1(c), P2 is P(a), P2(b), or P2(c), and P3 is P3(a), P3(b), or P3(c), TABLE-US-00007 P1(a) 5′-SH-C.sub.6H.sub.12-TTT TTT TTT ATA TAT ATA  (SEQ ID NO: 1)-3′ P1(b) 5′-SH-C.sub.10H.sub.20-TTT TTT TAT ATA TAT  (SEQ ID NO: 2)-3′ P1(c) 5′-SH-C.sub.5H.sub.10-TTT TTT TTT TTT CTA TAT  ATA (SEQ ID NO: 3)-3′ P2(a) 5′-TAA CAT AAT TAG GTC TTT TTT  (SEQ ID NO:4)-C.sub.6H.sub.12-NH.sub.2-3′ P2(b) 5′-TAT CAG TCT GAC TTT TTT  (SEQ ID NO: 5)-C.sub.8H.sub.16-NH.sub.2-3′ P2(c) 5′-TAG CAT ATT CTG GCA TTT TTT  (SEQ ID NO: 6)-C.sub.6H.sub.12-NH.sub.2-3′ P3(a) 5′-GAC CTA ATT ATG AAAAAAAAAAAA  TTA TAT ATA TAT (SEQ ID NO: 7)-3′ P3(b) 5′-GTC AGA CTG ATA AAAAAAAAAAAAAAAAAA  ATA TAT ATA (SEQ ID NO: 8)-3′ P3(c) 5′-TGC CAG AAT ATG AAAAAAAAA CTA   TAT ATA TAG (SEQ ID NO: 9)-3′.

5. The gold/quantum dot nanoprobe according to claim 1, wherein a molar ratio of the P1-AuPNs to the P2-QDs to the P3 is 1:(30 to 70):(100 to 300).

6. A method for detecting active ricin in a sample solution, comprising: (S1) causing a reaction between the gold/quantum dot nanoprobe of claim 1 with active ricin in each of a plurality of solutions, wherein the concentration of active ricin in each solution is known, and obtaining a relationship between the concentration of active ricin and fluorescence intensity at 575 nm (I.sub.575) by drawing a standard logarithmic curve between the concentration of active ricin and fluorescence intensity at 575 nm; and (S2) adding the gold/quantum dot nanoprobe of claim 1 into the sample solution, monitoring the fluorescence intensity at 575 nm of the sample solution, and determining the concentration of the active ricin in the sample solution according to the relationship obtained in step (S1).

7. A method for visually detecting active ricin in a sample, comprising a step of: adding the gold/quantum dot nanoprobe according to claim 1 into a sample solution, irradiating the sample solution with an ultraviolet light flashlight, and determining the presence of active ricin in the sample solution when an orange light from the sample solution is visually observed.

8. The gold/quantum dot nanoprobe according to claim 5, wherein the molar ratio of the P1-AuPNs to the P2-QDs to the P3 is 1:(40 to 60):(170 to 220).

9. The gold/quantum dot nanoprobe according to claim 5, wherein the molar ratio of the P1-AuPNs to the P2-QDs to the P3 is 1:(45 to 55):(190 to 210).

10. The gold/quantum dot nanoprobe according to claim 5, wherein the molar ratio of the P1-AuPNs to the P2-QDs to the P3 is 1:50:200.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram in which deoxynucleotide chains on an AuNPs/QDs nanoprobe form a double strand structure to link AuNPs and QDs.

(2) FIG. 2 is a schematic diagram in which after active ricin is added, fluorescence of the AuNPs/QDs is converted into a switch-on state from a quenched state.

(3) FIGS. 3A and 3B are schematic diagrams of enrichment of ricin. FIG. 3A shows a synthesis process of ricin capture agent magnetic beads (MB@P(C-H)-mAb.sub.ricin); and FIG. 3B is a schematic diagram in which elution is carried out after the MB@P(C-H)-mAb.sub.ricin carries out enrichment on the ricin.

(4) FIGS. 4A and 4B are transmission electron microscopy (TEM) diagrams of MB@P(C-H).

(5) FIG. 5 is an infrared spectrogram obtained by Fourier Transformation Infrared (FT-IR) spectrometry before and after P(C-H) modifies MB.

(6) FIG. 6 is comparison of fluorescence intensity background signals and signal response to ricin at different molar ratios of P1-AuPNs to P2-QDs to P3.

(7) FIGS. 7A-7C show transmission electron microscopy (TEM) diagrams of P1-AuPNs, P2-QDs and an AuPNs/QDs nanoprobe.

(8) FIG. 8 is a surface-enhanced Raman spectrum of P1-AuPNs and AuPNs/QDs.

(9) FIG. 9 is a fluorescence spectrum of P2-QDs, a mixture of P1-AuPNs and the P2-QDs, and AuPNs/QDs.

(10) FIGS. 10A and 10B are TEM diagrams 20 min after different concentration of ricin is added into AuPNs/QDs; FIG. 10A is a TEM diagram after 30 ng/mL of ricin is added; and FIG. 10B is a TEM diagram after 80 ng/mL of ricin is added.

(11) FIG. 11 is a fluorescence spectrum of an AuPNs/QDs nanoprobe buffer solution after active ricin with different concentrations (0 to 100 ng/mL) is added.

(12) FIG. 12 is a relationship diagram of a denary logarithm value of the concentration (ng/mL) of active ricin and fluorescence intensity of a characteristic peak of AuPNs/QDs at 575 nm.

(13) FIG. 13 is a comparison diagram of fluorescence intensity at 575 nm two hours after 20 ng/mL of active ricin and 500 ng/mL of interfering substances are added into an AuPNs/QDs nanoprobe solution.

(14) FIG. 14 is a comparison diagram of detection on active ricin by an AuPNs/QDs nanoprobe stored for 4 months and an AuPNs/QDs nanoprobe prepared in field.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(15) The present disclosure will be further illustrated in connection with the drawings and embodiments, the embodiments are implemented on the premise of the technical solutions of the present disclosure, the detail implementation mode and the specific operation process are given, but the scope of protection of the present disclosure is not limited to the following embodiments.

(16) Unless otherwise specified, methods and reagents used in the following embodiments are all conventional methods and conventional reagents.

(17) Single strand oligodeoxynucleotides (ssODN) are purchased from Shanghai Sangon Biotechnology Co. Ltd. and purified through high-performance liquid chromatography. The ssODN used in the present disclosure are listed as follows:

(18) Intact ricin with purity exceeding 95% by a SDS-PAGE test is extracted from castor beans according to the following standard procedure (Tang, J.; Xie, J.; Shao, N.; Yan, Y. Electrophoresis, 2006, 27, 1303-1311). The water-soluble core-shell QDs (the ZnS-capped CdSe QDs, modified with thioglycolic acid) are purchased from Wuhan Jayuan Quantum Dots Co. Ltd. Ricin beta monoclonal antibodies (mAb.sub.ricin) CP37 and CP75 are purchased from Thermo Scientific. Bis(p-sulfonatophenyl)phenyl phosphine dehydrate dipotassium salt (BSPP) is purchased from the Sigma-Aldrich. Deionized water is purified by a Milli-Q water purification system, and is used throughout this experiment. Sulfo-NHS is purchased from Thermo Scientific.

(19) Instruments used in the present disclosure are as shown below:

(20) The morphology of nanoparticles is observed with a transmission electron microscopy (TEM, JEM-2000EX, Japan).

(21) FT-IR spectra are tested by using Bruker Vertex 70.

(22) Powder X-ray diffraction (XRD) patterns use a product of the Rigaku smart lab (Rigaku, Japan) under the test condition of Cu Kα radiation (λ=1.5406 Å).

(23) Zeta potentials and hydrodynamic sizes are measured by Malvern Nanosizer, purchased from Malvern Instruments Ltd., United Kingdom.

(24) Magnetic properties of magnetic beads are measured by a Physical Property Measurement System (PPMS, Cryogenic, 12 Tesla).

(25) The UV-vis absorption spectra use Shimadzu 3600.

(26) The fluorescence spectra are tested by using Shimadzu RF-5301PC.

Embodiment 1 Preparation and Characterization of Ricin Capture Agent Magnetic Beads (MB@P(C-H)-mAb.SUB.ricin.)

Synthesis of 3-methacryloylaminoethyl-2-carboxyethyl-dimethylammonium betaine (carboxybetaine methacrylamide) (CBMAA)

(27) According to a previously published procedure (Banerjee, I., Pangule, R. C., Kane, R. S., Adv Mater 2011, 23,690-718). In detail, DMAEMA (19.4 g, 114 mmol) was dissolved in 100 mL of anhydrous THF in a round bottom flask under vigorous stirring and cooled to 0° C. Subsequently, β-propiolactone (11.5 g, 160 mmol) was dissolved in 30 mL of anhydrous THF and added dropwise under argon for a period of about 1 h. The reaction was allowed to proceed for 24 h at 4° C. in a refrigerator. The white precipitate was filtered off, washed with anhydrous THF and ether, and dried under high vacuum. The product was confirmed by .sup.1H NMR. The synthetic route is as follows:

(28) ##STR00001##

Synthesis of Propyl-4-(trimethoxysilyl)benzyl sulfocarbonate (carbonotrithioate) (CTA)

(29) Reversible addition fragmentation chain transfer (RAFT) initiator, propyl-4-(trimethoxysilyl) benzyl carbonotrithioate (CTA), was synthesized according to the literature [(Qu, Z., Hu, F., Chen, K., Duan, Z., Gu, H., Xu, H., J. Colloid and Interface Sci. 2013, 398, 82-87)]. In detail, 1-propanethiol (6.6 mmol) was charged into a stirred suspension of K3PO4 (1.02 g, 6.6 mmol) in anhydrous acetone (15 mL), followed by stirring for about half an hour. CS2 (1.1 mL, 18 mmol) was added and the solution turned to bright yellow. After stirring for another 10 min, (4-(chloromethyl)phenyl)-trimethoxysilane (1.43 mL, 6.6 mmol) was added and the mixture was then stirred at ambient temperature in nitrogen atmosphere for 13 h. The mixture was concentrated, diluted with dichloromethane and filtered off. After removing the solvent from the filtrate under reduced pressure the resulting yellow residue was purified by column chromatography on silica gel to yield a bright yellow oil. The product was confirmed by .sup.1HNMR.

Preparation of Magnetic beads MB@P(C-H)

(30) Copolymer brush grafted MBs (MB@P(C-H) were obtained by RAFT polymerization. In order to obtain Fe.sub.3O.sub.4@SiO.sub.2 (MBs) modified by an initiator CTA, 50 mg of CTA was added into 100 mL of 1.0 mg/mL magnetic bead absolute ethyl alcohol suspension, and reflux was carried out for 5 hours under the nitrogen. The obtained CTA-MB was collected and washed for three times with ethyl alcohol. Finally, the CTA-MB was suspended in ethyl alcohol for standby application.

(31) In order to obtain a copolymer of the CBMAA and HEMA, i.e., P(C-H) polymer brushes, so as to graft the polymer brushes onto the magnetic beads (MBs), 0.1 g of the standby CTA-MB, 20 mg of AIBN, 1.0 mL of HEMA and 0.3 g of CBMAA were dissolved in 10 mL of degassed water and methyl alcohol mixed solvent (1:1 (v/v)). After bubbling was carried out for 30 minutes with nitrogen, a reaction container was sealed and heated at the temperature of 80° C. After the reaction was performed for 5 hours, a product was diluted and washed for three times with DMF to obtain the MB@P(C-H).

(32) FIG. 4A is a transmission electron microscopy (TEM) diagram of MB@P(C-H), and FIG. 4B is a partial enlarged view. It can be distinguished that the diameter of Fe.sub.3O.sub.4 core is about 120 nm, and the SiO.sub.2 layer of about 10 nm, polymer brushes P(C-H) of 15-20 nm in thickness, respectively. As measured by DLS, the average hydrodynamic diameter of the MB@P(C-H) was 182 nm with the DPI of 0.24. It was consistent with TEM images.

(33) In order to further verify that the polymer brushes P(C-H) were really grafted onto the magnetic beads, FIG. 5 is an infrared spectrogram obtained by Fourier Transformation Infrared (FT-IR) spectrometry before and after P(C-H) modifies MB, and it can be seen from the figure that the MB@P(C-H) and the P(C-H) have similar FT-IR spectra. There are strong peaks around 1100 cm.sup.−1 assigned to Si—O bond which confirm the presence of SiO.sub.2 both in the MBs and the MB@P(C-H). As for the MB@P(C-H), the wide absorption band around 3405 cm.sup.−1 was attributed to the stretching vibration of —OH. Several bands around 2940-2990 cm.sup.−1 indicate the presence of the alkane groups. The amide bands locate at about 1653 and 1585 cm.sup.−1. The carboxyl stretching vibration of the —COOH appear at 1721 cm.sup.−1. In addition, the peak at 1386 cm.sup.−1 represent the C—O symmetric stretching band. All of these bands indicated the success graft of the P(C-H) on the MBs. It is shown that the MB@P(C-H) has a weight loss of 17% by TGA analysis, which is significantly higher than that of the MBs with less than the 3% weight loss. Moreover, the saturation magnetization of the MB@P(C-H) was measured as 34.5 emu g.sup.−1, and the magnetization curve exhibits symmetry and passes accurately through the origin, which ensure a facile separation and reusability of the MB@P(C-H) from sample matrices.

Preparation of Ricin Capture Agent Magnetic Beads (MB@P(C-H)-mAb.SUB.ricin.)

(34) The ricin capture agent magnetic beads (MB@P(C-H)-mAb.sub.ricin) were obtained by covalently linking a ricin monoclonal antibody (mAb.sub.ricin) and the MB@P(C-H). The ricin monoclonal antibody (mAb.sub.ricin) was covalently linked on the MB@P(C-H) via a typical EDC catalyzed amino-carboxyl coupling reaction. The specific steps were that: the MB@P(C-H) was firstly activated by N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) (10 mg/mL) and EDC (20 mg/mL) in 1.0 mL of 10 mM 2-(N-morpholine)ethanesulfonic acid (MES) buffer; after 30 minutes, a supernatant was removed; 10 mg of mAb.sub.ricin (CP37 or CP75) dispersed in a 10 mM PBS solution was added; and the reaction was performed at room temperature for 5 hours, and then the obtained product was allowed to stand overnight at the temperature of 4° C. to obtain the MB@P(C-H)-mAb.sub.ricin after the reaction was quenched by 0.5% glycine.

Embodiment 2 Capture Ability of Ricin Capture Agent Magnetic Beads (MB@P(C-H)-mAb.SUB.ricin.) for Ricin

(35) For extracting ricin, a series of standard solutions with the ricin concentrations ranged from 10 to 50 μg/mL was added to 1.0 mL, 50 mg/mL of ricin capture agent magnetic beads (MB@P(C-H)-mAb.sub.ricin) solution. After incubating 2 h, the equilibrium concentrations of ricin in the supernatants were determined by the BCA protein assay. In addition, the binding kinetic between ricin and the MB@P(C-H)-mAb.sub.ricin was examined by mixing 50 mg of the MB@P(C-H)-mAb.sub.ricin with 50 μg ricin in 1.0 mL PBS buffer. At different incubating times from 20 to 120 min, the concentrations of ricin in the supernatant were determined by the BCA protein assay. The equilibrium adsorption amount (Q) of the MB@P(C-H)-mAb.sub.ricin was calculated based on the equation below:
Q=(C.sub.0−C.sub.e).Math.V.Math.m.sup.−1.Math.10.sup.3(mg/g).

(36) Here, C.sub.0 (μg mL.sup.−1) represent the initial ricin concentration in PBS buffer; C.sub.e (μg mL.sup.−1) is the equilibrium concentration of ricin in the supernatant; V (mL) is the volume of sample solution; m (g) is the mass of the MB@P(C-H)-mAb.sub.ricin.

(37) It is found that each gram of the MB@P(C-H))-mAb.sub.ricin contained 28 mg monoclonal antibody mAb.sub.ricin, and each gram of the MBs contained 6.2 mg monoclonal antibody mAb.sub.ricin. It is demonstrated that polymer brushes P(C-H) have influence on magnetic beads capture agent. The MB@P(C-H)-mAb.sub.ricin reached to the saturated adsorption within 20 min, suggesting the flexible interface of the MB@P(C-H)-mAb.sub.ricin facilitated the recognition and binding between mAb.sub.ricin and ricin by decreasing steric hindrance, thus achieved fast adsorption balance. In order to achieve the full recovery rate, in the embodiment of the present disclosure, the time of capturing ricin by the MB@P(C-H)-mAb.sub.ricin was 1 h. Table 2 is a comparison of the active ricin capture abilities when two types of different magnetic beads, i.e., the MB@P(C-H)-Ab.sub.ricin prepared in the present disclosure and MB@P(ConA/Gal) previously prepared by the inventor (refer to the document Anal. Chem. 2017, 89, 12209-12216), were used as capture agents in complex matrices. Concentration of active ricin was tested by LC-MS/MS.

(38) TABLE-US-00004 TABLE 2 Concentration Concentration of Concentration of of added ricin captured by ricin captured by ricin MB@P(C-H)-mAb.sub.ricin MB@P(ConA/Gal) Sample (ng/mL) (ng/mL) (ng/mL) Diluted 5.0 4.1 2.1 Human 50.0 43.0 28.3 Serum Orange Juice 5.0 4.2 2.5 50.0 44.5 32.5 Ham 5.0 3.6 1.7 50.0 39.3 19.8 Sandwich 5.0 3.7 1.2 50.0 38.5 21.6 Milk 5.0 4.1 1.5 50.0 42.3 24.3 Coffee 5.0 4.3 3.2 50.0 45.6 37.6

(39) It can be seen from Table 2 that the capture agent MB@P(C-H)-mAb.sub.ricin provided by the present disclosure can efficiently capture the ricin in various complex and polluted matrices, the concentration of the ricin was measured by LC-MS/MS, and the recovery rate can achieve 72.0% to 86.0% (5 ng/mL) and 77.0% to 92% (50 ng/mL). Compared with the MB@P(ConA/Gal), the capture agent MB@P(C-H)-mAb.sub.ricin of the present disclosure also has good capture ability for the ricin even in the complex matrices due to the double effects of the anti-fouling capacity of the polymer brushes P(C-H) and the introduction of the ricin monoclonal antibody mAb.sub.ricin, thereby achieving the objective of detecting the trace amount of ricin.

Embodiment 3 Assembly and Characterization of Gold/Quantum Dot Nanoprobe (AuNPs/QDs)

(40) Single strand oligodeoxynucleotides (ssODN) used in this embodiment were P1 (a), P2 (a) and P3 (a) in Table 1, respectively.

(41) Spherical gold nanoparticles (AuNPs) were obtained by a seed growth method, the method can refer to the document (Bastús, N. G., Comenge, J., Puntes, V., Langmuir 2011, 27, 11098-11105), and the diameter was about 60 nm. After six times of growth, the concentration of AuNPs gel was 1.9×10.sup.11 (NP/mL). For stabilizing the gold nanoparticles, 15 mg of BSPP was added to 100 mL of gold nanoparticle sol under stirring and then a reaction was conducted for at least 10 hours. The obtained BSPP-AuNP sol was centrifuged, a supernatant was removed and then the obtained product was dispersed into 10 mL of ultrapure water. P1 modified gold nanoparticles (P1-AuNPs) were obtained via strong binding between gold particles and thiol, and the specific steps were that: 50 μL of 0.1 mmol/L P1 (a) was treated with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) of which the molar weight is 5 times of that of the P1 (a) for 1 hour at the temperature of 50° C. to reduce disulfide bonds, and then the treated P1 (a) was added into the BSPP-AuNP sol. In the incubation process, 0.1 mL of 0.1 mol/L NaCl solution was added every two hours. After 10 hours, P1 (a)-AuNPs were centrifuged at 8000 rpm for 10 minutes, and the supernatant was carefully removed.

(42) P2 modified quantum dots (P2-QDs) were prepared by using an EDC/sulfo-NHS cross-linking method, and the specific steps were that: firstly, commercially purchased quantum dots were purified by utilizing a hyperconcentration technology, and then the purified quantum dots were diluted to 1.0 μM with ultrapure water; the quantum dots were activated by sulfo-NHS (0.1 mg mL.sup.−1) and EDC (0.1 mg mL.sup.−1) in 1.0 mL of 1.0 mM MES buffer; after the reaction was performed for 30 minutes, P2 (a) (0.1 mmol/L, 100 μL) was added into the activated quantum dot solution, and 0.1M NaHCO.sub.3 was added to regulate pH to 8.5; the reaction was continued to be carried out for 10 hours at room temperature, the reaction was quenched by 0.5% glycine, and the obtained product was kept overnight at the temperature of 4° C.; and P2 (a)-QDs were purified by dextrangel-G50.

(43) Assembly of the AuNP/QDs nanoprobe was carried out in a buffer solution, and the buffer solution included 10 mM PBS with a pH of 7.4, 5 mM MgCl.sub.2 and 0.01% Tween 20. Generally, the P1 (a)-AuNPs, the P2 (a)-QDs and P3 (a) were mixed in a molar ratio of 1:50:200. The pairing hybridization reaction of bases was performed for 10 minutes at the temperature of 80° C., and then was performed for 2 hours at the temperature of 37° C. so as to obtain a fluorescence quenched AuNP/QD nanostructure probe. The AuNP/QD nanoprobe was subjected to centrifugal separation and purification, and was suspended in a 10 mM PBS buffer solution for later use.

(44) Fluorescence intensity background signals and signal response to ricin at different molar ratios of the P1-AuPNs to the P2-QDs to the P3 were tested, the molar ratios of the P1-AuPNs to the P2-QDs to the P3 were 1:50:100, 1:50:150, 1:50:200 and 1:50:250, respectively, detection results were shown in FIG. 6, and in comprehensive consideration of the detection sensitivity and the signal-to-noise ratio, the molar ratio of the P1-AuPNs to the P2-QDs to the P3 was 1:50:200.

(45) The prepared AuNPs/QDs nanoprobe was subjected to characterization. FIGS. 7A and 7B show transmission electron microscopy (TEM) diagrams of P1-AuPNs, P2-QDs and an AuPNs/QDs nanoprobe. FIG. 7A is a TEM diagram of the P1-AuNPs, and shows a spherical structure with the diameter of about 60 nm. The Zeta potential and the hydrodynamic diameter of the AuNPs unmodified by the P1 were −17.8 mV and 63 nm, respectively, and the Zeta potential and the hydrodynamic diameter of the P1 modified AuNPs were −26.5 mV and 68 nm, respectively. FIG. 7B is a TEM diagram of the P2-QDs, and shows a spherical structure with the average diameter of 6 nm. The Zeta potential and the hydrodynamic diameter of the QDs unmodified by the P2 were −23.7 mV and 10 nm, respectively, and the Zeta potential and the hydrodynamic diameter of the P2 modified QDs were −25.4 mV and 14 nm, respectively. The zeta potential of the AuNPs/QDs was −24.6 mV, while the hydrodynamic diameter was increased to 96.3 nm. FIG. 7C is a TEM diagram of a core-satellite structure of the AuNPs/QDs, and it can be seen from the figure that the bases on the single strand oligodeoxynucleotides (ssODN) P1, P2 and P3 were subjected to pairing hybridization to form double strand oligodeoxynucleotides (dsODN) so as to assemble the AuNPs and the QDs into the core-satellite structure. FIG. 8 is a surface-enhanced Raman spectrum of P1-AuPNs and AuPNs/QDs, a peak at 1,037 cm.sup.−1 showed wagging adsorption of a C—N bond, a peak at 1,320 cm.sup.−1 showed stretching adsorption of the C—N bond, a peak at 1,468 cm.sup.−1 showed stretching of COO.sup.−, and existence of the SERS peak above showed existence of the single strand oligodeoxynucleotides. FIG. 9 is a fluorescence spectrum of P2-QDs, a mixture of P1-AuPNs and the P2-QDs, and AuPNs/QDs. It can be obviously seen from the figure that after the QDs and the AuPNs formed the core-satellite structure through forming the double strand linkers, the distance between the QDs and the AuPNs was sufficiently small, and thus, a fluorescence quenching phenomenon was generated, so that the fluorescence characteristic peak of the AuNPs/QDs at 575 nm basically disappeared. An ideal energy receptor/donor was formed between the AuNPs and the QDs through fluorescence resonance energy transfer (FRET) so as to effectively quench fluorescence of the QDs. The phenomena all showed that the double strand structure was formed by pairing hybridization of the single strand oligodeoxynucleotides (ssODN) P1, P2 and P3 and the core-satellite structure of the AuNPs/QDs was assembled.

Embodiment 4 Detection on Active Ricin by Using AuNPs/QDs Nanoprobe

(46) An AuNP/QD nanoprobe (0.5 OD) reacted with active ricin with various concentrations (0, 10, 15, 20, 30, 40, 60, 80 and 100 ng/mL) in an ammonium acetate buffer solution (200 μL, 5 mmol/L, pH 4.0) in a 96-well plate. Incubation was carried out for 2 hours at the temperature of 38° C., and the fluorescence intensity of the solutions was tested. In order to monitor depurination reaction dynamics between the active ricin and the AuNP/QD nanoprobe (0.5 OD), the fluorescence values of the solutions were detected every 20 minutes, until the fluorescence intensity was stable.

(47) FIG. 10A is a TEM diagram after 30 ng/mL of active ricin is added into an AuNPs/QDs nanoprobe buffer solution, FIG. 10B is a TEM diagram after 80 ng/mL of active ricin is added into the AuNPs/QDs nanoprobe buffer solution, and particles in the circles in the figures were dissociated QDs. FIGS. 10A and 10B showed that when the concentration of the active ricin was gradually increased, more and more QDs were dissociated from AuNPs, so that the QDs were not quenched due to the fact that the QDs were away from the AuNPs. In consideration of the relationship between the specific depurination reaction of the active ricin and the reaction time as well as the concentration of the active ricin, in the monitoring process, 2 h was selected to be used as the reaction time, and then the fluorescence intensity was only related to the concentration of the ricin.

(48) FIG. 11 is a fluorescence spectrum of an AuPNs/QDs nanoprobe buffer solution after active ricin with different concentrations (0, 10, 15, 20, 30, 40, 60, 80 and 100 ng/mL) is added. It can be seen from the figure that the concentration of the added active ricin was gradually increased, and the fluorescence intensity of the characteristic peak of the AuNPs/QDs at 575 nm was gradually reduced. FIG. 12 is a relationship diagram of a denary logarithm value of the concentration of the active ricin and the fluorescence intensity of a characteristic peak of AuPNs/QDs at 575 nm, and a diagram of a Langmuir type is obtained. When the concentration of the active ricin exceeded 100 ng/mL, the curve showed nonlinearity, and probably because adsorption of the probe achieved a saturated state under higher concentration of the active ricin; and when the concentration of the active ricin was within a range of 10 to 100 ng/mL, the curve showed a good linear relationship. By the curve, the following formula can be obtained:
I.sub.575=634.8.Math.lgC−594.7(R.sup.2=0.995)

(49) Wherein I.sub.575 represents the fluorescence intensity of the characteristic peak of the AuNPs/QDs at 575 nm, C represents the concentration of the active ricin, and the unit is ng/mL. Therefore, the fluorescence intensity of a sample was detected, and then the concentration of the active ricin in the sample can be calculated by the formula above. The limit of detection obtained by the method was 7.46 ng/mL, and the value was calculated by a control signal with three times of a standard deviation. A relative standard deviation (RSD) of 6 times of parallel detection on 50 ng/mL of ricin was 5.4%, showing that the detection method of the present disclosure has very good repeatability.

(50) In order to detect anti-interference performance of the method of the present disclosure, the intensity of the fluorescence characteristic peak of the AuNPs/QDs at 575 nm in each interfering substance was tested. FIG. 13 is a comparison diagram of the fluorescence intensity at 575 nm two hours after 20 ng/mL of active ricin and 500 ng/mL of interfering substances are added into the AuPNs/QDs nanoprobe solution. It can be seen that common organic matters cannot generate a fluorescence switch-on effect on the AuNPs/QDs even under the high concentration (500 ng/mL), while the active ricin can generate a strong switch-on effect on the AuNPs/QDs nanoprobe provided by the present disclosure under the concentration of 20 ng/mL, so that the fluorescence intensity of the AuNPs/QDs at 575 nm was obviously improved and a very excellent anti-interference capacity was showed.

(51) Whether the ricin exists in the sample can also be qualitatively judged by judging whether the fluorescence is switched on. For example, the AuNPs/QDs nanoprobe provided by the present disclosure is added into the to-be-detected sample, after the sample is stabilized for a period of time, the sample is irradiated with an ultraviolet light flashlight in a dark place, and if the sample solution emits orange light, it is indicated that the active ricin with a certain concentration exists in the sample. That is because that if the active ricin exists in the sample, after the AuNPs/QDs nanoprobe is added, the fluorescence of the QDs can be switched on again, and at the moment, if the sample is irradiated with ultraviolet light, a photoluminescence phenomenon is generated, and light visible to naked eyes is generated. In addition, the AuNPs/QDs nanoprobe provided by the present disclosure has very high anti-interference capacity, and common organic impurities with high concentration also cannot generate a switch-on effect on the AuNPs/QDs nanoprobe. Therefore, by means of the AuNP/QD nanoprobe provided by the present disclosure, an effect of rapidly and visually detecting whether the ricin exists in the sample in field can be achieved only through the ultraviolet light flashlight without the help of other equipment.

Embodiment 5 Selective Detection on Ricin in Various Complex Matrices

(52) Preparation of Sample

(53) Various complex food and biological samples were used for detection so as to evaluate reliability of the detection method. The samples included lettuces, chicken, bread, fresh milk, coffee and human plasma. Coarse food and biological samples of the present disclosure were respectively prepared from diluted human serum, orange juice, coffee, fresh milk and sandwiches. A specific method included the steps: weighing a solid sample and diluting the solid sample with a PBS buffer solution, a volume-mass ratio of the PBS buffer solution to the solid sample being 1.0 mL/g; and after homogenizing for 5 min, centrifuging for 2 minutes at 1,200 rpm so as to remove large-volume residues. The human serum is diluted with the PBS buffer solution by 5 times. All the samples were filled with ricin with the specific concentration.

(54) Enrichment of Ricin

(55) 5 mg of MB@P(C-H)-mAb.sub.ricin and 1.0 mL of the sample solution containing the ricin were directly added into a test tube, and were continuously stirred and mixed for 1 hour. By adsorption of a magnet for magnetic beads, the magnetic beads capturing the ricin were enriched at the bottom of the test tube, a supernatant was poured out, the magnetic beads were eluted for 30 minutes with 100 μL of trifluoroacetic acid (TFA)/deionized water/ethyl alcohol (1:50:49, v/v/v) eluent at the temperature of 25° C. after being washed with PBST and PBS buffer solutions, then 0.9 mL of ammonium acetate buffer solution (5 mmol/L, pH 4.0) was added, and the ricin captured by the magnetic beads was released again.

(56) Detection of Ricin

(57) The ricin in the samples was detected by the AuNP/QD nanoprobe according to the method in Embodiment 4. In order to verify reliability of detection of active ricin by the AuNP/QD nanoprobe provided by the present disclosure, the ricin was also measured by LC-MS/MS as comparison. Detection results were shown as Table 3.

(58) TABLE-US-00005 TABLE 3 AuNPs/QDs nanoprobe method Concentration Monitoring LC-MS/MS of added ricin concentration Recovery method Samples (ng/mL) (ng/mL) rate (%) (ng/mL) Diluted 50.0 38.4 76.8 43.0 Human Serum Orange 50.0 39.5 79.0 44.5 Juice Ham 50.0 34.3 68.6 39.3 Sandwich 50.0 32.7 65.4 38.5 Milk 50.0 37.3 74.6 42.3 Coffee 50.0 39.6 79.2 45.2

(59) It can be seen from Table 3 that the AuNPs/QDs nanoprobe provided by the disclosure shows excellent accuracy and sensitivity when used for detection on the active ricin. The method based on the AuNPs/QDs nanoprobe provided by the present disclosure and the LC-MS/MS method obtain the basically consistent results, which shows reliability of the detection method provided by the present disclosure. A lethal dose LD50 of taking in the ricin is estimated as 3 mg/kg, and for a child with a weight of 35 kg, a lethal dose of taking in the ricin is 105 mg. By taking a beverage as an example, the lethal oral intake ricin concentration is about 0.4 mg/mL, and the limit of detection of the detection method using the AuNPs/QDs nanoprobe provided by the present disclosure is much less than the human body intake lethal dose in a general case. In the high-concentration active ricin sample, quantitative and semi-quantitative detection of the concentration of the active ricin can be carried out only by testing the fluorescence intensity at 575 nm after the AuNPs/QDs nanoprobe is added into the sample. In case that there is no need to clearly know the concentration of the ricin, just in the qualitative judgment of whether the ricin exists, the present disclosure provides a simpler method, i.e., a treated sample solution is added into an AuNPs/QDs nanoprobe solution, if the active ricin exists in the sample, the specific depurination reaction can be performed, then adenine on an AuNPs/QDs linker can be cut off so as to make the double strand linker loose, then QDs are dissociated from AuNPs, the fluorescence of the QDs is not quenched any more, at the moment, the sample solution is directly irradiated with an ultraviolet light flashlight, and due to photoluminescence, if the ricin exists in the sample, light visible to naked eyes can be emitted. Visual detection without large-sized equipment is implemented.

Embodiment 6 Storage Stability Test of AuNPs/QDs Nanoprobe

(60) In order to further detect whether a prepared AuNPs/QDs nanoprobe still has a detection effect on active ricin after being stored for a period of time, this embodiment was carried out. After the AuNPs/QDs nanoprobe was stored in a PBS buffer solution at the temperature of 4° C. for 1 month and 4 months, the fluorescence intensity of samples containing different concentrations of active ricin at 575 nm was detected according to the method in Embodiment 4 so as to obtain FIG. 14. After the AuNPs/QDs nanoprobe was stored for 4 months, a linear relationship formula between the concentration C (ng/mL) of the active ricin and the fluorescence intensity at 575 nm was that:
I.sub.575=628.4.Math.lgC−586.5(R.sup.2=0.993)

(61) It can be seen from FIG. 14 that after the AuNPs/QDs nanoprobe was stored for 1 month, the curve basically coincided with that before storage; and after the AuNPs/QDs nanoprobe was stored for 4 months, the curve was slightly deviated, but still can meet the requirement for detection on the active ricin.

Embodiment 7 Comparison of Various Methods for Detecting Active Ricin

(62) Table 4 listed some common methods for detecting active ricin. According to the method provided by the present disclosure, the active ricin was detected by means of the fluorescence quenched and switch-on process of AuNPs/QDs. Concentration of the active ricin can be known only by monitoring the fluorescence intensity of a sample without large-sized equipment. Tests prove that the method provided by the present disclosure is sensitive, reliable and short in time, and does not require large-sized instruments. In addition, in some special occasions, there is no need to know the specific concentration of the ricin, but it only needs to judge whether the ricin exists, and the present disclosure provides a visual method capable of carrying out detection in field, so that whether the ricin exists can be known only by one ultraviolet light flashlight through detecting whether the sample is in the fluorescence switch-on state, and an effective method is provided for rapid naked eye detection on the ricin.

(63) TABLE-US-00006 TABLE 4 Required Detection Instrument Limit of Methods Time Equipment Detection Samples Ref. Magnetic Bead Enrichment- nearly 4 h naked eyes or  7.46 diluted human serum, orange juice, the present Fluorescence Method spectrophotometer ng mL.sup.−1 ham, sandwich, milk and coffee disclosure Affinity Magnetic Enrichment nearly 4 h Naked eyes or ultraviolet- 12.5  juice, diluted human serum and Ref. 1 and Colorimetric Method visible spectrophotometer ng mL.sup.−1 drinking water Luminescence Analysis nearly 2 h spectrophotometer  0.8  buffer solution Ref. 2 ng mL.sup.−1 Real-Time Mass nearly 6 h mass spectrometer  5.7  buffer solution Ref. 3 Spectrometry Detection μg mL.sup.−1 Immunocapture and Mass nearly 4 h mass spectrometer  0.32 milk, juice, serum and saliva Ref. 4 Spectrometry Detection ng mL.sup.−1 Surface Enhanced nearly 3 h Raman spectrometer  8.9  juice, diluted human serum and Ref. 5 Raman Spectrometry ng mL.sup.−1 drinking water Immunocapture and Matrix- nearly 6 h matrix-assisted laser  0.2  buffer solution and milk Ref. 6 Assisted Laser Desorption desorption ionization ng mL.sup.−1 Ionization Time-of-Flight time-of-flight mass Mass Spectrometry specrometry Ref. 1: Sun, J., Wang, C., Shao, B., Wang, Z., Xue, D., Liu, Y., Qi, K., Yang, Y.; Niu, Y. Anal. Chem., 2017, 89, 12209-12216. Ref. 2: Sturm, M. B., Schramm, V. L., Anal. Chem., 2009, 81, 2847-2853. Ref. 3: Bevilacqua, V. H., Nilles, J. M., Rice, J. S., Connell, T. R., Schenning, A. M., Reilly, L. M., Durst, H. D., Anal. Chem., 2010, 82, 798-800. Ref. 4: Kalb, S. R., Barr, J. R., Anal. Chem. 2009, 81, 2037-2042. Ref. 5: Tang, J., Sun, J., Liu, R., Zhang, Z., Liu, J., Xie, J., ACS Applied Materials & Interfaces, 2016, 8, 2449-2455. Ref. 6: Wang, D., Baudys, J., Barr, J. R., Kalb, S. R., Anal. Chem., 2016, 88, 6867-6872.

(64) The specific embodiments above merely are used for schematically illustrating the contents of the present disclosure, but not intended to limit the contents of the present disclosure. What those skilled in the art can think of is that the specific structure in the present disclosure can have other change forms.