CN108304932A - The structure of logic gate based on silver nanoclusters and its application in intelligent measurement - Google Patents

Abstract

The invention belongs to the technical field of molecular logic gates of analytical chemistry, and relates to the establishment of a label-free, highly sensitive and highly selective miRNA intelligent detection method. The molecular logic gate is constructed based on silver nanoclusters and graphene oxide. The relative value of fluorescence intensity of silver nanoclusters is used as the judgment basis. When the relative fluorescence intensity is greater than 0.5, the output is "1". When the intensity is less than 0.5, the output is "0". The molecular logic gates include OR logic gates and INHIBIT logic gates, which can be used to check whether two different miRNAs (miR‑21 and miR‑141) exist respectively in actual samples. This logical detection method can identify the content of multiple target analytes in complex biological samples, and has potential application value in the early diagnosis of major diseases, multiple detection and clinical treatment.

Description

Construction of logic gates based on silver nanoclusters and its application in intelligent detection

technical field

The invention belongs to the technical field of molecular logic gates of analytical chemistry, and relates to the establishment of a label-free, highly sensitive and highly selective miRNA intelligent detection method.

Background technique

Molecular devices were proposed in the 1980s and mainly involve two fields: devices based on molecular scale and devices based on molecular materials. Among them, molecular-scale devices are currently one of the most active and widely researched hotspots. Molecular devices are crucial to the miniaturization of analytical chemistry instruments. Molecular devices have the characteristics of low cost and rapid response, which is conducive to the development of portable instruments to achieve real-time and online detection. Molecular devices mainly include molecular rectifiers, molecular memories, molecular motors, and molecular logic gates. This patent mainly uses molecular logic gates and biosensors to perform intelligent processing of analysis signals.

Due to the excellent molecular recognition ability of DNA, DNA-based molecular logic devices have achieved certain developments in the fields of disease diagnosis and biosensing. An important advantage of the DNA molecular logic gate is that it can generate a response signal to a low concentration of the target, thereby completing the detection. However, there are few label-free logic sensors based on the molecular level, which is due to the complex construction process of logic sensors that need to combine multiple logic gates. This kind of logic sensor can realize multiple detection of different targets in the same sample, which is of great significance for promoting the application of molecular logic gates in analytical chemistry. However, to date, only a few examples of molecular logic devices have achieved multiplexed detection of target molecules. We used graphene oxide and silver nanoclusters to construct a multiple logic gate based on OR and INHIBIT, and realized the multiple intelligent detection of two different miRNAs (miR-21 and miR-141) for the first time. In contrast, it can detect the presence of multiple analytes and perform complex logic analysis.

Contents of the invention

The purpose of the present invention is to provide a label-free multiple molecular logic gate based on silver nano-clusters and graphene oxide and the application of the logic device in multiple intelligence detection of miRNA. The OR and INHIBIT cascaded multiple logic gates are combinatorial logic circuits used to test whether two different miRNAs (miR-21 and miR-141) respectively exist in actual samples. This logical detection method can identify the content of multiple target analytes in complex biological samples.

The present invention is achieved in this way, a combination logic circuit for identifying whether two different miRNAs (miR-21 and miR-141) in actual samples exist respectively, specifically comprises the following steps:

1. Preparation of DNA-stabilized silver nanoclusters: the silver nanocluster template Ag-DNA1 or Ag-DNA2 was dissolved in a sodium phosphate buffer solution, heated to 90° C. for annealing, and then cooled to room temperature. Add 1mM silver ions into the solution, shake it well, let it stand for 30min, then add NaBH ₄ to reduce the silver ion, shake vigorously again for 1min, and store in the dark overnight. After the reaction was complete, a pale yellow DNA-protected silver nanocluster solution was obtained.

2. Construction of a molecular logic platform based on silver nanoclusters and graphene oxide: Silver nanoclusters protected by 100nM Ag-DNA1 and 100nMAg-DNA2 were mixed with 15μg/mL graphene oxide for 15 minutes as a logic operation platform.

3. Construction of OR logic gates: Based on the molecular logic platform, different combinations of miR-21 and miR-141 were added to construct OR logic gates. When nucleic acid sequences (miR-21 and miR-141) were added, the input was recorded as "1", otherwise "0". Based on whether the relative fluorescence intensity value is greater than 0.5 or not, when the relative fluorescence intensity is greater than 0.5, the output is "1", and when the relative fluorescence intensity is less than 0.5, the output is "0". miR-21 and miR-141 can build an OR logic gate on the molecular logic platform. When either value of miR-21 or miR-141 was input, the output signal was "1".

4. Construction of INHIBIT logic gates based on miR-21 and P1: Based on the molecular logic platform, adding different combinations of miR-21 and P1 to construct INHIBIT logic gates. miR-21 and P1 can build an INHIBIT logic gate on a molecular logic platform. When P1 was input, the output signal was "0", regardless of whether miR-21 was input at the same time in the system. Only when miR-21 is imported into the system but not P1, the output signal is "1".

5. Construction of INHIBIT logic gates based on miR-141 and P2: Based on the molecular logic platform, different combinations of miR-141 and P2 were added to construct INHIBIT logic gates. miR-141 and P2 can build an INHIBIT logic gate on a molecular logic platform. When P2 was input, the output signal was "0" regardless of whether miR-141 was input at the same time in the system. The output signal is "1" only when miR-141 is imported into the system but not P2.

6. Multivariate detection of miRNA: In order to determine whether there are two different miRNAs (miR-21 and miR-141) in the sample, we divided the same sample into four. First, four identical samples were added to the GO/Ag-DNA1/Ag-DNA2 molecular logic platform, which were recorded as sample 1, sample 2, sample 3, and sample 4. For the first sample, no other inputs are added. For the second sample, 1 μM P1 was added. For the third sample, 1 μM P2 was added. For the fourth sample, 1 μM P1 and 1 μM P2 were added. Output "1" and "0" according to the relative fluorescence intensity values of samples 1, 2, 3, and 4, and use the truth table to judge whether miR-21 and miR-141 exist in the samples.

Description of drawings

Figure 1 (A) Schematic diagram of OR logic gate construction. (B) Fluorescence spectra of OR logic gates. (C) Histogram of relative fluorescence intensity of OR logic gate. (D) The truth table of the OR logic gate;

Fig. 2 (A) Schematic diagram of the construction of INHIBIT logic gates based on miR-21 and P1. (B) Fluorescence spectra of miR-21 and P1-based INHIBIT logic gates. (C) Histogram of relative fluorescence intensity of miR-21 and P1-based INHIBIT logic gates. (D) the truth table of the INHIBIT logic gate;

Fig. 3 (A) Schematic diagram of the construction of INHIBIT logic gates based on miR-141 and P2. (B) Fluorescence spectra of miR-141 and P2-based INHIBIT logic gates. (C) Histogram of relative fluorescence intensity of miR-141 and P2-based INHIBIT logic gates. (D) the truth table of the INHIBIT logic gate;

Fig. 4 (A) Schematic diagram of INHIBIT-OR cascade logic gates for intelligent detection of miR-21 and miR-141. (B) Truth table for INHIBIT-OR cascade logic gates. "\" means that this situation can be ignored;

Detailed ways

The present invention will be further described below in conjunction with the examples, and the examples of the present invention are only used to illustrate the technical solution of the present invention, not to limit the present invention.

Example 1

Preparation of silver nanoclusters: Dissolve 5 μM Ag-DNA1 or Ag-DNA2 in sodium phosphate buffer solution (10mMNa ₂ HPO ₄ /NaH ₂ PO ₄ , 100mM CH ₃ COONa, 5mM Mg(CH ₃ COO) ₂ , pH 7.5) , then heated at 90 °C for 10 min, then cooled slowly to room temperature. Then add 30 μM AgNO ₃ to 5 μM Ag-DNA1 or Ag-DNA2 solution, mix well, and place the mixed solution in the dark for 30 minutes, so that the Ag ⁺ ions can fully interact with the C base. Finally, add 30 μM NaBH ₄ solution to the buffer system again and shake vigorously for 1 minute. The fluorescent silver nanoclusters were prepared by reacting overnight at 4°C in the dark.

Example 2

Construction of OR logic gates based on silver nanoclusters and graphene oxide: Based on the molecular logic platform, different combinations of miR-21 and miR-141 were added to construct OR logic gates. When nucleic acid sequences (miR-21 and miR-141) were added, the input was recorded as "1", otherwise "0". Based on whether the relative fluorescence intensity value is greater than 0.5 or not, when the relative fluorescence intensity is greater than 0.5, the output is "1", and when the relative fluorescence intensity is less than 0.5, the output is "0". When there is no input, the fluorescence of silver nanoclusters is quenched by graphene oxide, and the relative fluorescence intensity is less than 0.5, which is recorded as "0" as the output. Since the sequences of miR-21 and Ag-DNA1 are partially complementary, when miR-21 is added, Ag-DNA1 will hybridize with miR-21 to form a DNA/RNA double-stranded structure, which will desorb from the surface of graphene oxide . Therefore, the silver nanoclusters protected by Ag-DNA1 will also leave the surface of graphene oxide, and the fluorescence intensity is enhanced, and the relative fluorescence intensity is greater than 0.5, which is recorded as "1" as the output. Similarly, since the sequences of miR-141 and Ag-DNA2 are partially complementary, when miR-141 is added, Ag-DNA2 will hybridize with miR-141 to form a DNA/RNA double-stranded structure, which will be formed from graphene oxide. Surface desorption. Therefore, the silver nanoclusters protected by Ag-DNA2 will also leave the surface of graphene oxide, and the fluorescence intensity is enhanced, and the relative fluorescence intensity is greater than 0.5, which is recorded as the output as "1". Of course, when the two exist at the same time, the silver nanoclusters protected by Ag-DNA1 and the silver nanoclusters protected by Ag-DNA2 will desorb from the surface of graphene oxide, and the relative fluorescence intensity is greater than 0.5, which is recorded as "1". Thus, miR-21 and miR-141 can construct an OR logic gate on the molecular logic platform. When the platform inputs any value of miR-21 or miR-141, the output signal is "1".

Example 3

Construction of INHIBIT logic gates based on miR-21 and P1: Based on the molecular logic platform, different combinations of miR-21 and P1 were added to construct INHIBIT logic gates. When nucleic acid sequences (miR-21 and P1) were added, the input was recorded as "1", otherwise "0". Based on whether the relative fluorescence intensity value is greater than 0.5 or not, when the relative fluorescence intensity is greater than 0.5, the output is "1", and when the relative fluorescence intensity is less than 0.5, the output is "0". When there is no input, the fluorescence of silver nanoclusters is quenched by graphene oxide, and the relative fluorescence intensity is less than 0.5, which is recorded as "0" as the output. Since the sequences of miR-21 and Ag-DNA1 are partially complementary, when miR-21 is added, Ag-DNA1 will hybridize with miR-21 to form a DNA/RNA double-stranded structure, which will desorb from the surface of graphene oxide . Therefore, the silver nanoclusters protected by Ag-DNA1 will also leave the surface of graphene oxide, and the fluorescence intensity is enhanced, and the relative fluorescence intensity is greater than 0.5, which is recorded as "1" as the output. However, P1, Ag-DNA1, and Ag-DNA2 sequences do not have enough complementary bases, so when P1 is added, P1 and other DNA will not hybridize, so that the silver nanoclusters cannot leave the surface of graphene oxide, and the fluorescence signal remains unchanged. If the relative fluorescence intensity is less than 0.5, record the output as "0". Of course, when miR-21 and P1 exist at the same time, since miR-21 and P1 are completely complementary, miR-21 and P1 will preferentially hybridize, so that Ag-DNA1 cannot hybridize with miR-21. In this way, the silver nanoclusters also cannot leave the graphene oxide surface, the fluorescence signal remains unchanged, and the relative fluorescence intensity is less than 0.5, which is recorded as "0" as the output. Thus, miR-21 and P1 can construct an INHIBIT logic gate on the molecular logic platform.

Example 4

Construction of INHIBIT logic gates based on miR-141 and P2: Based on the molecular logic platform, different combinations of miR-141 and P2 were added to construct INHIBIT logic gates. When nucleic acid sequences (miR-141 and P2) were added, the input was recorded as "1", otherwise "0". Based on whether the relative fluorescence intensity value is greater than 0.5 or not, when the relative fluorescence intensity is greater than 0.5, the output is "1", and when the relative fluorescence intensity is less than 0.5, the output is "0". When there is no input, the fluorescence of silver nanoclusters is quenched by graphene oxide, and the relative fluorescence intensity is less than 0.5, which is recorded as "0" as the output. Since the sequences of miR-141 and Ag-DNA2 are partially complementary, when miR-141 is added, Ag-DNA2 will hybridize with miR-141 to form a DNA/RNA double-stranded structure, which will desorb from the surface of graphene oxide . Therefore, the silver nanoclusters protected by Ag-DNA2 will also leave the surface of graphene oxide, and the fluorescence intensity is enhanced, and the relative fluorescence intensity is greater than 0.5, which is recorded as the output as "1". However, P2, Ag-DNA1, and Ag-DNA2 sequences do not have enough complementary bases, so when P2 is added, P2 will not hybridize with other DNA, so that the silver nanoclusters cannot leave the surface of graphene oxide, and the fluorescence signal remains unchanged. If the relative fluorescence intensity is less than 0.5, record the output as "0". Of course, when miR-141 and P2 exist at the same time, since miR-141 and P2 are completely complementary, miR-141 and P2 will preferentially hybridize, so that Ag-DNA2 cannot hybridize with miR-141. In this way, the silver nanoclusters also cannot leave the graphene oxide surface, the fluorescence signal remains unchanged, and the relative fluorescence intensity is less than 0.5, which is recorded as "0" as the output. Thus, miR-141 and P2 can construct an INHIBIT logic gate on the molecular logic platform.

The OR and INHIBIT logic gates constructed in the examples are applied to the intelligent detection of multiple miRNAs in the same sample. The specific operation methods and results are as follows:

Application example 1

In order to be able to determine whether there are two different miRNAs (miR-21 and miR-141) in the sample, we need to use the INHIBIT-OR cascade logic gate to complete the signal output. First, we divided the same sample (original sample) into four. Then, four identical samples (original samples) will be added to the GO/Ag-DNA1/Ag-DNA2 molecular logic platform respectively, which will be recorded as sample 1, sample 2, sample 3, and sample 4. For the first sample, no other inputs are added. For the second sample, 1 μM P1 was added. For the third sample, 1 μM P2 was added. For the fourth sample, 1 μM P1 and 1 μM P2 were added. When the first sample has no obvious fluorescent signal, we can consider that miR-21 and miR-141 do not exist in the original sample, and the test ends. If the first sample produces a fluorescent signal, it means that the original sample contains at least one of miR-21 and miR-141. This allows us to perform a fluorescence test on the second sample. If the second one does not produce a fluorescent signal, it means that the original sample contains miR-21. This is because the second sample has more P1 than the first sample, so that the miR-21 in the original sample is inhibited and cannot be detected. Hybridization produces a fluorescent signal. If the second one still has a fluorescent signal, it means that the original sample contains at least miR-141. We need to further test whether the original sample only contains miR-141. This requires a fluorescence test on the third sample. If the third sample does not produce a fluorescent signal, it means that the original sample only contains miR-141. This is because the third sample added more P2 than the first sample, so that the miR-141 in the original sample was inhibited by P2 and could not hybridize to generate fluorescent signals. If the third sample still has fluorescent signals, it means that the original sample contains both miR-21 and miR-141 miRNAs. In this way, only when P1 and P2 are added at the same time, the fluorescence signal will be quenched (see the fourth sample). See Figure 4B for the specific truth table. This detection method combined with logic provides a good conceptual model for the intelligent detection aspect of analytical chemistry.

Claims (4)

1. a kind of structure of logic gate of silver nanoclusters and its application in intelligent measurement, which is characterized in that the molecule Logic gate is that fluorescent quenching occurs after being contacted based on silver nanoclusters and graphene oxide, and hybridization forms the original of fluorescence recovery after double-strand Manage it is built-up, using the fluorescence intensity relative value of silver nanoclusters as basis for estimation, when relative intensity of fluorescence be more than 0.5 when, Output is " 1 ", when relative intensity of fluorescence is less than 0.5, is exported as " 0 ".The Molecular Logic Gates include OR logic gates and Cascaded logic door that INHIBIT logic gates and the two are connected in series simultaneously is applied to logic detection.

2. the construction method and logic detection application of OR logic gates as described in claim 1 and INHIBIT logic gates, special Sign is as follows：

(1) OR logic gates：By the silver nanoclusters of 100nM Ag-DNA1 and 100nM Ag-DNA2 protections, stone is aoxidized with 15 μ g/mL Black alkene is used as logical operation platform after mixing 15 minutes.Believe using RNA chain miR-21 and miR-141 as input according to shown in Fig. 1 Number, the fluorescence intensity of silver nanoclusters builds OR logic gates as output signal.

(2) the INHIBIT logic gates based on miR-21 and P1：It is molten with silver nanoclusters used in (1) and graphene oxide mixing Liquid as molecular logic platform, according to shown in Fig. 2 using RNA/DNA chain miR-21 and P1 as input signal, fluorescence intensity conduct Output signal builds INHIBIT logic gates.

(3) the INHIBIT logic gates based on miR-141 and P2：It is molten with silver nanoclusters used in (1) and graphene oxide mixing Liquid as molecular logic platform, according to shown in Fig. 3 using RNA/DNA chain miR-141 and P2 as input signal, fluorescence intensity conduct Output signal builds INHIBIT logic gates.

(4) the multivariable detection of miRNA：Judge two kinds whether are respectively present in sample using INHIBIT-OR cascaded logic doors Different miRNA (miR-21 and miR-141) exports " 1 " and " 0 " according to the relative intensity of fluorescence value of sample, is judged with truth table MiR-21 and miR-141 whether there is in sample.

(5) in the structure of the logic gate as described in claim 1 and 2, required DNA sequence dna is as follows：

Ag-DNA1:TGAACATCAGACTGATAACCTAATCCCCCCCCCCCC

Ag-DNA2:CGATCTTTACCTGACAGTCTTA ATCCCCCCCCCCCC

P1:TCAACATCAGTCTGATAAGCTA

P2:CCATCTTTACCAGACAGTGTTA

miR-21:UAGCUUAUCAGACUGAUGUUGA

miR-141:UAACACUGUCUGGUAAAGAUGG。

3. in the structure of logic gate as described in claim 1, input oligonucleotide chain (miR-21, miR-141, P1 and P2) Concentration and Ag-DNA1/Ag-DNA2 concentration ratios are 10:1.

4. the logic detection work(to miRNA may be implemented in the molecular logic device based on silver nanoclusters as described in claim 1 Energy.