Molecular logic gate sensing system for intelligently identifying antibiotics

Abstract

The invention belongs to the field of intelligent recognition of biosensors, and relates to a molecular logic gate sensing system for intelligently recognizing antibiotics. According to the invention, antibiotics KAN and STR are taken as targets for the first time, a molecular logic gate sensing system for intelligently identifying antibiotics is constructed, the molecular logic gate sensing system comprises an AND molecular logic gate, an XOR molecular logic gate and an INHIBIT molecular logic gate, and intelligent identification of KAN and STR is realized. The molecular logic gate sensing system is simple to construct and low in cost, a reaction element is pure nucleic acid, protease, an antibody and a nano material are not used, a separation and purification process is not needed, and on-site rapid intelligent recognition of antibiotics can be realized in combination with a visual sensing result.

Description

Technical Field

[0001] This invention belongs to the field of intelligent biosensor recognition, specifically involving a set of molecular logic gate sensing systems for intelligent recognition of antibiotics. Background Technology

[0002] Kanamycin (KAN) and streptomycin (STR) are broad-spectrum amino acid glycoside antibiotics that exhibit good antibacterial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, and can be used to treat or inhibit infections caused by pathogenic microorganisms. They play an important role in disease treatment, livestock farming, and food preservation. However, the overuse of antibiotics can easily lead to drug resistance in pathogenic bacteria and seriously endanger environmental health and food safety. Therefore, the identification of these two antibiotics is of great significance.

[0003] Conventional chemical detection methods and conventional biosensor methods are insufficient for the intelligent identification of these two antibiotics. Molecular logic gate-based biosensors can simulate the binary operation principle of computers, encoding the analyte with 0s and 1s. Through molecular operations and truth tables, intelligent identification and sensing of the analyte can be achieved. In this invention, we have, for the first time, constructed a molecular logic gate sensing system for intelligent antibiotic identification based on nucleic acid aptamer recognition, DNA strand displacement reaction, and G-tetramer colorimetry, using KAN and STR as target analytes. This system includes AND, XOR, and INHIBIT molecular logic gates, enabling intelligent identification and sensing of these two antibiotics at the molecular level. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an intelligent molecular logic gate sensing system for recognizing two antibiotics (KAN and STR). The system involves the recognition of antibiotics by nucleic acid aptamers, nucleic acid probe strand displacement reaction, and G-tetramer colorimetric reaction. It combines binary encoding with AND, XOR, and INHIBIT molecular logic gates to achieve intelligent recognition of antibiotics.

[0005] The first aspect of the present invention is to provide a set of probes for detecting kanamycin and / or streptomycin.

[0006] A second aspect of the present invention is to provide a biosensor.

[0007] A third aspect of the present invention is to provide a reagent kit.

[0008] The fourth aspect of this invention aims to provide a detection system for kanamycin and / or streptomycin.

[0009] The fifth aspect of this invention aims to provide the use of the probe set described in the first aspect of this invention, the biosensor described in the second aspect of this invention, the reagent kit described in the third aspect of this invention, and / or the detection system described in the fourth aspect of this invention in the preparation of products for detecting kanamycin and / or streptomycin.

[0010] The sixth aspect of this invention aims to provide a method for detecting the coexistence of kanamycin and streptomycin using AND molecular logic gates.

[0011] The seventh aspect of this invention aims to provide a method for detecting either kanamycin or streptomycin using an XOR molecular logic gate.

[0012] The objective of the eighth aspect of this invention is to provide a method for detecting the presence of kanamycin but the absence of streptomycin using the INHIBIT molecular logic gate.

[0013] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is: In a first aspect, the present invention provides a set of probes for detecting kanamycin and / or streptomycin, said probe set comprising DNA1, DNA2, DNA3, DNA4, and DNA5.

[0014] In some embodiments of the present invention, the DNA1 includes kanamycin aptamer sequences a, b, and c.

[0015] In some embodiments of the present invention, the DNA2 is partially complementary to the nucleic acid aptamer sequence a and sequence b. In some embodiments of the present invention, the DNA3 includes sequence e, complementary sequence b*, and streptomycin aptamer sequence d.

[0016] In some embodiments of the present invention, the DNA4 is partially complementary to the nucleic acid aptamer sequence d and the complementary sequence b*.

[0017] In some embodiments of the present invention, the DNA5 includes complementary sequence c*, complementary sequence e*, G-tetramer sequence f, and sequence e.

[0018] In some embodiments of the present invention, b and b* are complementary pairings, c and c* are complementary pairings, and e and e* are complementary pairings.

[0019] In some embodiments of the present invention, the probe set for detecting kanamycin and / or streptomycin includes any one of 1) to 3): 1) AND molecular logic gate probe set; 2) XOR molecular logic gate probe set; 3) INHIBIT molecular logic gate probe set.

[0020] In these logic gate sensors, antibiotics KAN and STR are encoded with 0 and 1, respectively. 0 indicates that KAN or STR is not present in the detection system; 1 indicates that KAN or STR is present in the detection system. (0,0) indicates that KAN and STR are not present; (1,0) indicates that only KAN is present; (0,1) indicates that only STR is present; (1,1) indicates that both KAN and STR are present.

[0021] In some embodiments of the present invention, the sequence of the AND molecular logic gate probe set is as follows: DNA1: 5'-TGGGGGTTGAGGCTAAGCCGA(a)-TTCGCGATCG(b)-CACATA(c)-3' (SEQ ID NO: 1); where region a is the aptamer sequence of kanamycin; region b has 10 bases and region c has 6 bases.

[0022] DNA2: 5'-CGCGAATCGGCTTAGC-3' (SEQ ID NO: 2); 6 bases are complementary to the b region of DNA1, and 10 bases are complementary to the a region of DNA1.

[0023] DNA3: 5'-ACTGAACTAG(e)-CGATCGCGAA(b*)-GGGGTCTGGTGTTCTGCTTTGTTCTGTCGGGTCGT(d)-3' (SEQ ID NO: 3); where the d region is the nucleic acid aptamer sequence of streptomycin; the b* region has 10 bases and the e region has 10 bases.

[0024] DNA4: 5'-ACCAGACCCCTTCGCG-3' (SEQ ID NO: 4); 6 bases are complementary to the b* region of DNA3, and 10 bases are complementary to the d region of DNA3.

[0025] DNA5: 5'-TATGTG(c*)-CTAGTTCAGT(e*)-GGGTAGGGCGGGTTGGG)(f)-ACTGAACTAG(e)-3' (SEQ ID NO: 5); where the c* region has 6 bases; the e* region and the e region are complementary, each with 10 bases; and the f region has 17 bases, forming a G-tetramer sequence.

[0026] The detection principle of AND molecular logic gates is as follows: Figure 1 As shown: The AND molecular logic gate contains five DNA probes: DNA1, DNA2, DNA3, DNA4, and DNA5. DNA1 and DNA2 hybridize to form a DNA1-DNA2 complex. DNA3 and DNA4 hybridize to form a DNA3-DNA4 complex. DNA5 is a stem-loop DNA probe, where the f region is the loop portion, which is a G-tetrameric nucleic acid sequence rich in G bases.

[0027] In the (0,0) state, the reaction system contains no KAN and STR. At this time, the sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0028] In the (1,0) state, KAN binds to the a region of DNA1 (the a region is the nucleic acid aptamer sequence of KAN), thereby replacing DNA2 and forming a DNA1-KAN complex. This complex cannot open the stem-loop structure of DNA5. At this time, the sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0029] In the (0,1) state, STR binds to the d region of DNA3 (the d region is the nucleic acid aptamer sequence of STR), thereby replacing DNA4 and forming a DNA3-STR complex. This complex cannot open the stem-loop structure of DNA5. At this time, the sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0030] In the (1,1) state, KAN binds to region a in DNA1; STR binds to region d in DNA3. Further, DNA1-KAN binds to DNA3-STR via bb* complementation, thus bringing region c in DNA1 and region e in DNA3 closer together. Subsequently, the ce region hybridizes via c*-e* complementation in DNA5, opening the stem-loop structure of DNA5 and exposing region f. Region f is a G-tetrameric nucleic acid sequence that binds to heme to form G-tetrameric-heme deoxyribonuclease. G-tetrameric-heme deoxyribonuclease exhibits highly efficient peroxidase catalytic activity, catalyzing the conversion of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (TMB+), resulting in an observable blue color change in the reaction solution. The solution exhibits an absorbance at 650 nm higher than 0.1, and the signal output is 1.

[0031] In the process of intelligent identification of KAN and STR by the AND logic gate biosensor, the signal output can only be 1 when both antibiotics are present at the same time; in other combinations, the output signal is 0.

[0032] In some embodiments of the present invention, the sequence of the XOR molecular logic gate probe group is as follows: DNA1: 5'-TGGGGGTTGAGGCTAAGCCGA(a)-TTCGCGATCG(b)-CACATA(c)-3' (SEQ ID NO: 1); where region a is the aptamer sequence of kanamycin; region b has 10 bases and region c has 6 bases.

[0033] DNA2: 5'-CGCGAATCGGCTTAGC-3' (SEQ ID NO: 2); 6 bases are complementary to the b region of DNA1, and 10 bases are complementary to the a region of DNA1.

[0034] DNA3: 5′-AACTAG(e)-CGATCGCGAA(b*)-GGGGTCTGGTGTTCTGC TTTGTTCTGTCGGGTCGT(d)-3′ (SEQ ID NO: 6); where the d region is the nucleic acid aptamer sequence of streptomycin; the b* region has 10 bases and the e region has 6 bases.

[0035] DNA4: 5'-ACCAGACCCCTTCGCG-3' (SEQ ID NO: 4); 6 bases are complementary to the b* region of DNA3, and 10 bases are complementary to the d region of DNA3.

[0036] DNA5: 5'-TATGTG(c*)-CGATCGCGAA(b*)-GGGTAGGGCGGGTTGGG(f)-TTCGCGATCG(b)-CTAGTT(e*)-3' (SEQ ID NO: 7); wherein the c* region has 6 bases; the b* region is complementary to the b region and both have 10 bases; the f region has 17 bases and is a G-tetramer sequence; the e* region has 6 bases.

[0037] The detection principle of XOR molecular logic gates is as follows: Figure 2 As shown: The XOR molecular logic gate contains five DNA probes: DNA1, DNA2, DNA3, DNA4, and DNA5. DNA1 and DNA2 hybridize to form a DNA1-DNA2 complex. DNA3 and DNA4 hybridize to form a DNA3-DNA4 complex. DNA5 is a stem-loop DNA probe, where the f region is the loop portion, which is a G-tetrameric nucleic acid sequence rich in G bases.

[0038] In the (0,0) state, the reaction system contains no KAN and STR. At this time, the sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0039] In the (1,0) state, the reaction system contains the antibiotic KAN. KAN binds to region a in DNA1 (region a is the aptamer sequence of KAN), thereby displacing DNA2 and exposing the complete region b. Only then can region bc hybridize complementary to the b*-c* region in DNA5, opening the stem-loop structure of DNA5 and exposing region f. Region f is the nucleic acid sequence of a G-tetramer, which binds to heme to form G-tetramer-heme deoxyribonuclease. G-tetramer-heme deoxyribonuclease has highly efficient peroxidase catalytic activity, catalyzing the conversion of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (TMB+). This results in an observable blue color change in the reaction solution, with an absorbance of 0.1 at 650 nm and a signal output of 1.

[0040] In the (0,1) state, the reaction system contains the antibiotic STR. At this point, STR binds to the d region of DNA3 (the d region is the aptamer sequence of STR), thereby displacing DNA4 and exposing the complete b* region. Only then can the b*-e region hybridize complementaryly with the be* region of DNA5, opening the stem-loop structure of DNA5 and exposing the f region. The f region is the nucleic acid sequence of the G-tetramer, which binds to heme to form G-tetramer-heme deoxyribonuclease. G-tetramer-heme deoxyribonuclease exhibits highly efficient peroxidase catalytic activity, catalyzing the conversion of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (TMB+). This results in an observable blue color change in the reaction solution, with an absorbance at 650 nm higher than 0.1 and a signal output of 1.

[0041] In the (1,1) state, the reaction system contains both antibiotics KAN and STR. KAN binds to region a of DNA1, thereby displacing DNA2 and exposing the complete region b. STR binds to region d of DNA3, thereby displacing DNA4 and exposing the complete region b*. At this point, the exposed regions b and b* hybridize complementaryly, forming a DNA1-DNA3 complex. This complex cannot open the stem-loop structure of DNA5, and region f remains enclosed in the loop portion of DNA5. Therefore, it cannot form a catalytically active G-tetramer-heme deoxyribonuclease with Hemin. In this state, the sensing system does not react, the reaction solution is colorless, the UV absorbance at 650 nm is less than 0.1, and the output signal is 0.

[0042] In the process of intelligent recognition of KAN and STR by the XOR molecular logic gate biosensor, when the two antibiotics are present alone, they can generate a high output signal, with the signal output being 1; when there is no antibiotic or the two antibiotics are present at the same time, a low output signal is generated, with the signal output being 0.

[0043] In some embodiments of the present invention, the sequence of the INHIBIT molecular logic gate probe set is as follows: DNA1: 5'-TGGGGGTTGAGGCTAAGCCGA(a)-TTCGCGATCG(b)-CACATA(c)-3' (SEQ ID NO: 1); where region a is the aptamer sequence of kanamycin; region b has 10 bases and region c has 6 bases.

[0044] DNA2: 5'-CGCGAATCGGCTTAGC-3' (SEQ ID NO: 2); 6 bases are complementary to the b region of DNA1, and 10 bases are complementary to the a region of DNA1.

[0045] DNA3: 5'-CACATACCGT(c)-ATCGCGAA(b*)-GGGGTCTGGTGTTCTGCTTTGTTCTGTCGGGTCGT(d)-3' (SEQ ID NO: 8); where region d is the nucleic acid aptamer sequence of streptomycin; region b* has 8 bases and region c has 10 bases.

[0046] DNA4: 5'-ACCAGACCCCTTCGCGAT-3' (SEQ ID NO: 9); 8 bases are complementary to the b* region of DNA3, and 10 bases are complementary to the d region of DNA3. DNA5: 5'-CACATACCGT(c)-GGGTAGGGCGGGTTGGG(f)-ACGGTATGTG(c*)-ATCGCGAA(b*)-3' (SEQ ID NO: 10); wherein the b* region has 8 bases; the c* region is complementary to the c region and both have 10 bases; the f region has 17 bases and is a G-tetramer sequence.

[0047] The detection principle of INHIBIT molecular logic gates is as follows: Figure 3 As shown: The INHIBIT molecular logic gate contains five DNA probes: DNA1, DNA2, DNA3, DNA4, and DNA5. DNA1 and DNA2 hybridize to form a DNA1-DNA2 complex. DNA3 and DNA4 hybridize to form a DNA3-DNA4 complex. DNA5 is a stem-loop DNA probe, where the f region is the loop portion, which is a G-tetrameric nucleic acid sequence rich in G bases.

[0048] In the (0,0) state, the reaction system contains no KAN and STR. At this time, the sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0049] In the (1,0) state, KAN binds to region a in DNA1 (region a is the aptamer sequence of KAN), thereby displacing DNA2 and forming the DNA1-KAN complex, exposing the complete region b. Only then can region bc hybridize complementaryally with the b*-c* region in DNA5, opening the stem-loop structure of DNA5 and exposing region f. Region f is the nucleic acid sequence of the G-tetramer, which binds to heme to form G-tetramer-heme deoxyribonuclease. G-tetramer-heme deoxyribonuclease has highly efficient peroxidase catalytic activity, catalyzing the conversion of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) into a blue product (TMB+), thus allowing observation of a blue change in the reaction solution. The solution has an absorbance value higher than 0.1 at 650 nm, and the signal output is 1.

[0050] In the (0,1) state, STR binds to the d region of DNA3 (the d region is the nucleic acid aptamer sequence of STR), thereby replacing DNA4 and forming a DNA3-STR complex. At this time, the exposed b*-c region cannot open the stem-loop structure of DNA5. The sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0051] In the (1,1) state, KAN binds to the a region of DNA1; STR binds to the d region of DNA3. Furthermore, DNA1-KAN binds to DNA3-STR through bb* complementarity. At this point, the complex cannot open the stem-loop structure of DNA5. The sensing system does not react, the reaction solution is colorless, the UV absorbance of the reaction solution at 650 nm is less than 0.1, and the output signal is 0.

[0052] In the intelligent identification of KAN and STR by the INHIBIT logic gate biosensor, the signal output can only be 1 when KAN is present alone; in other combinations, the output signal is 0.

[0053] A second aspect of the present invention provides a biosensor comprising the probe set described in the first aspect of the present invention.

[0054] In some embodiments of the present invention, the biosensor includes a reaction buffer.

[0055] A third aspect of the present invention is to provide a reagent kit.

[0056] In some embodiments of the present invention, the kit includes a reaction buffer, heme, and a colorimetric reagent.

[0057] In some embodiments of the present invention, the colorimetric reagent includes 3,3',5,5'-tetramethylbenzidine and H2O2.

[0058] A fourth aspect of the present invention provides a detection system for kanamycin and / or streptomycin, the detection system comprising the following components: 1) Detection components for kanamycin and / or streptomycin; 2) Data processing components; 3) Result output components.

[0059] In some embodiments of the present invention, the detection component for kanamycin and / or streptomycin includes the probe set described in the first aspect of the present invention, the biosensor described in the second aspect of the present invention, and / or the kit described in the third aspect of the present invention.

[0060] In some embodiments of the present invention, the detection component includes a detection instrument selected from at least one of an ELISA reader and a quantitative fluorescence meter.

[0061] A fifth aspect of the present invention provides the use of the probe set described in the first aspect of the present invention, the biosensor described in the second aspect of the present invention, the reagent kit described in the third aspect of the present invention, and / or the detection system described in the fourth aspect of the present invention in the preparation of products for detecting kanamycin and / or streptomycin.

[0062] In some embodiments of the present invention, the product includes a biosensor and a detection kit.

[0063] A sixth aspect of the present invention provides a method for detecting the coexistence of kanamycin and streptomycin using AND molecular logic gates. This method includes the following steps: 1) Mix and incubate DNA1 and DNA2 from the AND molecular logic gate probe set, and mix and incubate DNA3 and DNA4 to obtain DNA1-DNA2 complex and DNA3-DNA4 complex. 2) Mix the DNA1-DNA2 complex, the DNA3-DNA4 complex, and DNA5 from the AND molecular logic gate probe set to obtain a mixture; 3) Add the test sample to the mixture to react, and obtain reaction solution 1; 4) Add heme to reaction solution 1 and react to obtain reaction solution 2; 5) Add 3,3',5,5'-tetramethylbenzidine and H2O2 to reaction solution 2, carry out the reaction, and detect the results; If the result is positive, the sample contains kanamycin and streptomycin.

[0064] A seventh aspect of the present invention provides a method for detecting either kanamycin or streptomycin using an XOR molecular logic gate, comprising the following steps: 1) Mix and incubate DNA1 and DNA2 from the XOR molecular logic gate probe set, and mix and incubate DNA3 and DNA4 to obtain DNA1-DNA2 complex and DNA3-DNA4 complex. 2) Mix the DNA1-DNA2 complex, the DNA3-DNA4 complex, and DNA5 from the XOR molecular logic gate probe set to obtain a mixture; 3) Add the test sample to the mixture to react, and obtain reaction solution 1; 4) Add heme to reaction solution 1 and react to obtain reaction solution 2; 5) Add 3,3',5,5'-tetramethylbenzidine and H2O2 to reaction solution 2, carry out the reaction, and detect the results; If the result is positive, the sample contains kanamycin and streptomycin.

[0065] An eighth aspect of the present invention provides a method for detecting the presence of kanamycin but the absence of streptomycin using the INHIBIT molecular logic gate, comprising the following steps: 1) Mix DNA1 and DNA2 from the INHIBIT molecular logic gate probe set and incubate them together, and mix DNA3 and DNA4 and incubate them together to obtain DNA1-DNA2 complex and DNA3-DNA4 complex. 2) Mix the DNA1-DNA2 complex, DNA3-DNA4 complex, and DNA5 from the INHIBIT molecular logic gate probe set to obtain a mixture; 3) Add the test sample to the mixture to react, and obtain reaction solution 1; 4) Add heme to reaction solution 1 and react to obtain reaction solution 2; 5) Add 3,3',5,5'-tetramethylbenzidine and H2O2 to reaction solution 2, carry out the reaction, and detect the results; If the result is positive, the sample contains kanamycin and streptomycin.

[0066] In some embodiments of the present invention, the reaction concentration of DNA1-5 is 10-1000 nM.

[0067] In some embodiments of the present invention, the incubation time is 5 to 30 minutes.

[0068] In some embodiments of the present invention, the reaction time is 5 to 60 minutes.

[0069] In some embodiments of the present invention, the concentration of heme is 100~500 nm.

[0070] In some embodiments of the present invention, the temperature of the mixing incubation and reaction is room temperature.

[0071] The beneficial effects of this invention are: This invention, for the first time, constructs an intelligent molecular logic gate sensing system for antibiotic identification, targeting the antibiotics kanamycin (KAN) and streptomycin (STR). This system includes AND, XOR, and INHIBIT molecular logic gates, enabling intelligent identification of KAN and STR. Using kanamycin (KAN) and streptomycin (STR) as the two target antibiotics, nucleic acid aptamers are used to identify the two antibiotics, triggering a DNA strand substitution reaction. A G-tetramer is used as a signal reporter probe, and antibiotic identification is achieved by observing the color change of the reaction solution. Antibiotics are encoded using 0 and 1, where 0 indicates the absence of KAN or STR in the detection system, and 1 indicates their presence. The output signal is defined as 1 when the reaction solution turns blue or the UV absorbance is greater than 0.1; otherwise, it is defined as 0. In the AND molecular logic gate, only the input combination (1,1) produces a signal output of 1. In the XOR molecular logic gate, only the input combinations (1,0) and (0,1) produce a signal output of 1. In the INHIBIT molecular logic gate, only the input combination of (1,0) produces a signal output of 1.

[0072] This molecular logic gate sensing system is simple to construct and low in cost. The reaction element is pure nucleic acid, without the use of proteases, antibodies, or nanomaterials. It does not require separation and purification processes. Combined with visualized sensing results, it can achieve rapid and intelligent on-site identification of antibiotics. Attached Figure Description

[0073] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram illustrating the principle of AND molecular logic gates for intelligent antibiotic recognition.

[0074] Figure 2 This is a schematic diagram illustrating the principle of intelligent antibiotic recognition using XOR molecular logic gates.

[0075] Figure 3 This is a schematic diagram illustrating the principle of INHIBIT's intelligent recognition of antibiotics using molecular logic gates.

[0076] Figure 4 The results of the AND molecular logic gate detection include: (A) Visual response analysis of the AND molecular logic gate to KAN and STR; (B) UV absorption spectra of the AND molecular logic gate to KAN and STR; (C) UV absorbance intensity of the AND molecular logic gate to KAN and STR; (D) Truth table of the AND molecular logic gate; and (E) Circuit diagram of the AND molecular logic gate.

[0077] Figure 5 The results of XOR molecular logic gate detection include: (A) Visual response analysis of XOR molecular logic gate to KAN and STR; (B) UV absorption spectra of XOR molecular logic gate to KAN and STR; (C) UV absorbance intensity of XOR molecular logic gate to KAN and STR; (D) Truth table of XOR molecular logic gate; and (E) Circuit diagram of XOR molecular logic gate.

[0078] Figure 6 The results of the INHIBIT molecular logic gate detection include: (A) Visual response analysis of the INHIBIT molecular logic gate to KAN and STR; (B) UV absorption spectra of the INHIBIT molecular logic gate to KAN and STR; (C) UV absorbance intensity of the INHIBIT molecular logic gate to KAN and STR; (D) Truth table of the INHIBIT molecular logic gate; and (E) Circuit diagram of the INHIBIT molecular logic gate. Detailed Implementation

[0079] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0080] Example 1: Design of DNA Probe Nucleic Acid Sequences Using AND Molecular Logic Gates In this embodiment, DNA1, DNA2, DNA3, DNA4, and DNA5 were prepared, as detailed below: DNA1: 5'-TGGGGGTTGAGGCTAAGCCGA(a)-TTCGCGATCG(b)-CACATA(c)-3' (SEQ ID NO: 1); where region a is the aptamer sequence of kanamycin; region b has 10 bases and region c has 6 bases.

[0081] DNA2: 5'-CGCGAATCGGCTTAGC-3' (SEQ ID NO: 2); 6 bases are complementary to the b region of DNA1, and 10 bases are complementary to the a region of DNA1.

[0082] DNA3: 5'-ACTGAACTAG(e)-CGATCGCGAA(b*)-GGGGTCTGGTGTTCTGCTTTGTTCTGTCGGGTCGT(d)-3' (SEQ ID NO: 3); where the d region is the nucleic acid aptamer sequence of streptomycin; the b* region has 10 bases and the e region has 10 bases.

[0083] DNA4: 5'-ACCAGACCCCTTCGCG-3' (SEQ ID NO: 4); 6 bases are complementary to the b* region of DNA3, and 10 bases are complementary to the d region of DNA3.

[0084] DNA5: 5'-TATGTG(c*)-CTAGTTCAGT(e*)-GGGTAGGGCGGGTTGGG)(f)-ACTGAACTAG(e)-3' (SEQ ID NO: 5); where the c* region has 6 bases; the e* region and the e region are complementary, each with 10 bases; and the f region has 17 bases, forming a G-tetramer sequence.

[0085] Example 2: Intelligent Recognition Results of KAN and STR by AND Molecular Logic Gate Biosensor 1. Detection Method DNA1-5 from Example 1 were tested using the following method: (1) All nucleic acid probes were dissolved in buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.4).

[0086] (2) Mix 100 nM DNA1 with 100 nM DNA2 and incubate at room temperature for 15 minutes to form DNA1-DNA2 complex.

[0087] (3) Mix 100 nM DNA3 with 100 nM DNA4 and incubate at room temperature for 15 minutes to form DNA3-DNA4 complex.

[0088] (4) DNA5 was prepared as a 100 nM solution.

[0089] (5) Mix the nucleic acids prepared in steps (2) to (4) and set aside.

[0090] (6) According to the four combinations of molecular logic gate inputs: (0,0); (1,0); (0,1); (1,1), KAN or STR is added in step (5) respectively: (0,0) means neither KAN nor STR is added; (1,0) means only 1 nM KAN is added; (0,1) means only 1 nM STR is added; (1,1) means both 1 nM KAN and 1 nM STR are added. After adding KAN or STR according to the four combinations, the reaction is carried out at room temperature for 30 minutes.

[0091] (7) Add 200 nM heme to the reaction solution in step (6) and react at room temperature for 20 minutes.

[0092] (8) Add 0.01% of 3,3',5,5'-tetramethylbenzidine (TMB) solution and 5% H2O2 solution to the reaction solution in step (7). After reacting at room temperature for 15 minutes, observe the color change of the solution and record the ultraviolet absorbance at 650 nm.

[0093] 2. Experimental Results Experimental results are as follows Figure 4 As shown.

[0094] When KAN and STR are absent from the reaction system, i.e., in the (0,0) state, the reaction solution is colorless, and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0095] When the reaction system contains only KAN, i.e., in the (1,0) state, the reaction solution is colorless, and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0096] When the reaction system contains only STR, i.e., in the (0,1) state, the reaction solution is colorless, and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0097] When both KAN and STR are present in the reaction system, i.e., in the (1,1) state, the reaction solution turns blue, and the signal output is 1. The reaction solution has a high UV absorbance value at 650 nm, which is higher than 0.1, and the signal output is 1.

[0098] Example 3: XOR molecular logic gate DNA probe nucleic acid sequence design In this embodiment, DNA1, DNA2, DNA3, DNA4, and DNA5 were prepared, as detailed below: DNA1: 5'-TGGGGGTTGAGGCTAAGCCGA(a)-TTCGCGATCG(b)-CACATA(c)-3' (SEQ ID NO: 1); where region a is the aptamer sequence of kanamycin; region b has 10 bases and region c has 6 bases.

[0099] DNA2: 5'-CGCGAATCGGCTTAGC-3' (SEQ ID NO: 2); 6 bases are complementary to the b region of DNA1, and 10 bases are complementary to the a region of DNA1.

[0100] DNA3: 5′-AACTAG(e)-CGATCGCGAA(b*)-GGGGTCTGGTGTTCTGC TTTGTTCTGTCGGGTCGT(d)-3′ (SEQ ID NO: 6); where the d region is the nucleic acid aptamer sequence of streptomycin; the b* region has 10 bases and the e region has 6 bases.

[0101] DNA4: 5'-ACCAGACCCCTTCGCG-3' (SEQ ID NO: 4); 6 bases are complementary to the b* region of DNA3, and 10 bases are complementary to the d region of DNA3.

[0102] DNA5: 5'-TATGTG(c*)-CGATCGCGAA(b*)-GGGTAGGGCGGGTTGGG(f)-TTCGCGATCG(b)-CTAGTT(e*)-3' (SEQ ID NO: 7); wherein the c* region has 6 bases; the b* region is complementary to the b region and both have 10 bases; the f region has 17 bases and is a G-tetramer sequence; the e* region has 6 bases.

[0103] Example 4: Intelligent Recognition Results of KAN and STR by XOR Molecular Logic Gate Biosensor 1. Detection Method DNA1-5 from Example 3 were tested using the following method: (1) All nucleic acid probes were dissolved in buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.4).

[0104] (2) Mix 100 nM DNA1 with 100 nM DNA2 and incubate at room temperature for 15 minutes to form DNA1-DNA2 complex.

[0105] (3) Mix 100 nM DNA3 with 100 nM DNA4 and incubate at room temperature for 15 minutes to form DNA3-DNA4 complex.

[0106] (4) DNA5 was prepared as a 100 nM solution.

[0107] (5) Mix the nucleic acids prepared in steps (2) to (4) and set aside.

[0108] (6) According to the four combinations of molecular logic gate inputs: (0,0); (1,0); (0,1); (1,1), KAN or STR is added in step (5) respectively: (0,0) means neither KAN nor STR is added; (1,0) means only 1 nM KAN is added; (0,1) means only 1 nM STR is added; (1,1) means both 1 nM KAN and 1 nM STR are added. After adding KAN or STR according to the four combinations, the reaction is carried out at room temperature for 30 minutes.

[0109] (7) Add 200 nM heme to the reaction solution in step (6) and react at room temperature for 20 minutes.

[0110] (8) Add 0.01% of 3,3',5,5'-tetramethylbenzidine (TMB) solution and 5% H2O2 solution to the reaction solution in step (7). After reacting at room temperature for 15 minutes, observe the color change of the solution and record the ultraviolet absorbance at 650 nm.

[0111] 2. Experimental Results Experimental results are as follows Figure 5 As shown.

[0112] When KAN and STR are absent from the reaction system, i.e., in the (0,0) state, the reaction solution is colorless, and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0113] When the reaction system contains only KAN, i.e., in the (1,0) state, the reaction solution turns blue, and the signal output is 1. The reaction solution has a high UV absorbance value at 650 nm, which is higher than 0.1, and the signal output is 1.

[0114] When the reaction system contains only STR, i.e., in the (0,1) state, the reaction solution turns blue, and the signal output is 1. The reaction solution has a high UV absorbance value at 650 nm, which is higher than 0.1, and the signal output is 1.

[0115] When both KAN and STR are present in the reaction system, i.e., in the (1,1) state, the reaction solution is colorless and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0116] Example 5: INHIBIT Molecular Logic Gate DNA Probe Nucleic Acid Sequence Design In this embodiment, DNA1, DNA2, DNA3, DNA4, and DNA5 were prepared, as detailed below: DNA1: 5'-TGGGGGTTGAGGCTAAGCCGA(a)-TTCGCGATCG(b)-CACATA(c)-3' (SEQ ID NO: 1); where region a is the aptamer sequence of kanamycin; region b has 10 bases and region c has 6 bases.

[0117] DNA2: 5'-CGCGAATCGGCTTAGC-3' (SEQ ID NO: 2); 6 bases are complementary to the b region of DNA1, and 10 bases are complementary to the a region of DNA1.

[0118] DNA3: 5'-CACATACCGT(c)-ATCGCGAA(b*)-GGGGTCTGGTGTTCTGCTTTGTTCTGTCGGGTCGT(d)-3' (SEQ ID NO: 8); where region d is the nucleic acid aptamer sequence of streptomycin; region b* has 8 bases and region c has 10 bases.

[0119] DNA4: 5'-ACCAGACCCCTTCGCGAT-3' (SEQ ID NO: 9); 8 bases are complementary to the b* region of DNA3, and 10 bases are complementary to the d region of DNA3. DNA5: 5'-CACATACCGT(c)-GGGTAGGGCGGGTTGGG(f)-ACGGTATGTG(c*)-ATCGCGAA(b*)-3' (SEQ ID NO: 10); wherein the b* region has 8 bases; the c* region is complementary to the c region and both have 10 bases; the f region has 17 bases and is a G-tetramer sequence.

[0120] Example 6: Intelligent Recognition Results of KAN and STR by the INHIBIT Molecular Logic Gate Biosensor 1. Detection Method DNA1-5 from Example 5 were tested using the following method: (1) All nucleic acid probes were dissolved in buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.4).

[0121] (2) Mix 100 nM DNA1 with 100 nM DNA2 and incubate at room temperature for 15 minutes to form DNA1-DNA2 complex.

[0122] (3) Mix 100 nM DNA3 with 100 nM DNA4 and incubate at room temperature for 15 minutes to form DNA3-DNA4 complex.

[0123] (4) DNA5 was prepared as a 100 nM solution.

[0124] (5) Mix the nucleic acids prepared in steps (2) to (4) and set aside.

[0125] (6) According to the four combinations of molecular logic gate inputs: (0,0); (1,0); (0,1); (1,1), KAN or STR is added in step (5) respectively: (0,0) means neither KAN nor STR is added; (1,0) means only 1 nM KAN is added; (0,1) means only 1 nM STR is added; (1,1) means both 1 nM KAN and 1 nM STR are added. After adding KAN or STR according to the four combinations, the reaction is carried out at room temperature for 30 minutes.

[0126] (7) Add 200 nM heme to the reaction solution in step (6) and react at room temperature for 20 minutes.

[0127] (8) Add 0.01% of 3,3',5,5'-tetramethylbenzidine (TMB) solution and 5% H2O2 solution to the reaction solution in step (7). After reacting at room temperature for 15 minutes, observe the color change of the solution and record the ultraviolet absorbance at 650 nm.

[0128] 2. Experimental Results Experimental results are as follows Figure 6 As shown.

[0129] When KAN and STR are absent from the reaction system, i.e., in the (0,0) state, the reaction solution is colorless, and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0130] When the reaction system contains only KAN, i.e., in the (1,0) state, the reaction solution turns blue, and the signal output is 1. The reaction solution has a high UV absorbance value at 650 nm, which is higher than 0.1, and the signal output is 1.

[0131] When the reaction system contains only STR, i.e., in the (0,1) state, the reaction solution is colorless, and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0132] When both KAN and STR are present in the reaction system, i.e., in the (1,1) state, the reaction solution is colorless and the signal output is 0. The UV absorbance of the reaction solution at 650 nm is low, below 0.1, and the signal output is 0.

[0133] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A probe set for detecting kanamycin and / or streptomycin, characterized in that: the probe set comprises DNA1, DNA2, DNA3, DNA4 and DNA5; the DNA1 comprises a nucleic acid aptamer sequence a, a sequence b and a sequence c of kanamycin; the DNA2 is partially complementary to the nucleic acid aptamer sequence a and the sequence b; the DNA3 comprises a sequence e, a complementary sequence b* and a nucleic acid aptamer sequence d of streptomycin; the DNA4 is partially complementary to the nucleic acid aptamer sequence d and the complementary sequence b*; the DNA5 comprises a complementary sequence c*, a complementary sequence e*, a G-quadruplex sequence f and the sequence e. 2.The probe set of claim 1, characterized in that: when the probe set is used for detecting kanamycin and streptomycin, the sequences of the DNA1-DNA5 are shown in SEQ ID NO: 1-5, respectively. 3.The probe set of claim 1, characterized in that: when the probe set is used for detecting the presence of only one of kanamycin or streptomycin, the sequences of the DNA1-DNA5 are shown in SEQ ID NO: 1, 2, 6, 4, 7, respectively. 4.The probe set of claim 1, characterized in that: when the probe set is used for detecting the presence of kanamycin and the absence of streptomycin, the sequences of the DNA1-DNA5 are shown in SEQ ID NO: 1, 2, 8, 9, 10, respectively. 5.A biosensor comprising the probe set of any one of claims 1-4. 6.A detection system for kanamycin and / or streptomycin, comprising the following components: 1) a detection component for kanamycin and / or streptomycin; 2) a data processing component; 3) a result output component; the detection component for kanamycin and / or streptomycin comprises the probe set of any one of claims 1-4 and / or the biosensor of claim 5. 7.Use of the probe set of any one of claims 1-4, the biosensor of claim 5 and / or the detection system of claim 6 in the preparation of a product for detecting kanamycin and / or streptomycin. 8.A method for detecting the presence of both kanamycin and streptomycin using an AND molecular logic gate, comprising the following steps: 1) incubating DNA1 and DNA2 of claim 2 together, and incubating DNA3 and DNA4 together to obtain DNA1-DNA2 complex and DNA3-DNA4 complex; 2) mixing the DNA1-DNA2 complex, the DNA3-DNA4 complex and the DNA5 of claim 2 to obtain a mixture; 3) adding a detection sample to the mixture to obtain a reaction liquid 1; 4) adding hematin to the reaction liquid 1 to obtain a reaction liquid 2; 5) adding 3,3',5,5'-tetramethylbenzidine and H2O2 to the reaction liquid 2 to obtain a detection result; if the result is positive, the detection sample contains kanamycin and streptomycin. 9.A method for detecting the presence of only one of kanamycin or streptomycin using an XOR molecular logic gate, comprising the following steps: 1) incubate DNA1 and DNA2, DNA3 and DNA4 as claimed in claim 3 to obtain DNA1-DNA2 complex, DNA3-DNA4 complex; 2) mix DNA1-DNA2 complex, DNA3-DNA4 complex and DNA5 as claimed in claim 3 to obtain a mixture; 3) add the sample to be detected to the mixture to obtain reaction liquid 1; 4) add hematin to reaction liquid 1 to obtain reaction liquid 2; 5) add 3,3',5,5'-tetramethylbenzidine and H2O2 to reaction liquid 2 to obtain the detection result; If the result is positive, it indicates that the sample contains kanamycin but not streptomycin, or the sample contains streptomycin but not kanamycin.

10. A method for detecting the presence of kanamycin but not streptomycin using INHIBIT molecular logic gate, comprising the following steps: 1) incubate DNA1 and DNA2, DNA3 and DNA4 as claimed in claim 3 to obtain DNA1-DNA2 complex, DNA3-DNA4 complex; 2) mix DNA1-DNA2 complex, DNA3-DNA4 complex and DNA5 as claimed in claim 3 to obtain a mixture; 3) add the sample to be detected to the mixture to obtain reaction liquid 1; 4) add hematin to reaction liquid 1 to obtain reaction liquid 2; 5) add 3,3',5,5'-tetramethylbenzidine and H2O2 to reaction liquid 2 to obtain the detection result; If the result is positive, it indicates that the sample contains kanamycin but not streptomycin.

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