CN112326957A - Ratio type DNA molecular machine for biological analysis - Google Patents

Ratio type DNA molecular machine for biological analysis Download PDF

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CN112326957A
CN112326957A CN201910643084.3A CN201910643084A CN112326957A CN 112326957 A CN112326957 A CN 112326957A CN 201910643084 A CN201910643084 A CN 201910643084A CN 112326957 A CN112326957 A CN 112326957A
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吕弋
王超群
刘睿
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Sichuan University
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Abstract

The invention discloses a ratio type DNA molecular machine which can be used for biological analysis. The DNA molecular machine is triggered by target molecules, and walking of the target molecules is realized through a 'bridge combustion' mechanism, so that biological signals are converted and amplified into mass spectrum signal ratios. In short, the target molecule-activated DNA molecular machinery releases large amounts of gold nanoparticles to become in solution197The Au signal greatly increased in a short time. By simultaneous detection of target-dependent molecules197Au and internal standard unrelated to target molecule115In and calculating the ratio of the signals generated by the In to quantitatively analyze the target. The invention combines the high sensitivity of the DNA molecular machine method and the high accuracy and the ratiometric ICPMS analysisStability and the like. Under optimal conditions, the detection limit for the level of fM for analysis of the target molecule was obtained. In addition, the invention provides the idea of developing a ratiometric DNA molecular machine that accurately detects multiple molecules simultaneously.

Description

Ratio type DNA molecular machine for biological analysis
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to the fields of ratio type DNA molecular machines, nucleic acid detection methods and stable isotope (Metal stable isotope) sensing.
Background
The 2016 Nobel prize awarded to 3 scientists studying artificial molecular machines, a emerging technology that has attracted considerable attention from researchers. Among them, DNA molecules have strong sequence programmability and precise molecular recognition ability, are ideal intelligent nanomaterials, and are considered as important elements for designing molecular machines. The design and application of DNA molecular machine become important research field and hot spot of current DNA nanometer technology. Among them, the DNA molecular machine itself has a signal amplification function, which has a significant advantage in bioanalysis, and is a molecular machine that can travel "continuously" along a precisely designed one-, two-, or three-dimensional DNA track, thereby generating a cascade signal enhancement that converts biological signal amplification into a detectable signal. After these pioneering efforts, one important challenge is to guide accurate and reliable quantification.
The ratio sensor calculates the intensity ratio of two signals by measuring the two signals simultaneously for analysis, and the internal standard signal correction can obviously improve the accuracy, the reproducibility and the stability of the method. In addition to the above, inorganic element mass spectrometry is an excellent accurate analysis tool, can be used for ratio measurement and has been widely used for metal stable isotope labeling in recent years.
Disclosure of Invention
Technical problem to be solved
In order to achieve accurate and reliable quantification of nucleic acid molecules, the present invention provides a ratio-type DNA molecular machine and a method for nucleic acid detection using the same. The molecular machine activated by the target molecule releases a large amount of AuNP-labeled DNA chain fragments, so that197The ICPMS signal of Au increases greatly in a short time. At the same time, the elements115In was analyzed as an Internal Standard (IS) to correct measurement errors of ICPMS and sample variations In sample preparation. The method combines the advantages of high sensitivity of a DNA molecular machine, high accuracy of ratio measurement, high stability of ICPMS and the like. Under optimal conditions, detection of fM levels for analysis of target molecules is obtainedAnd (4) limiting.
(II) technical scheme
In order to solve the above problems, a first object of the present invention is to provide a rate-type DNA molecular machine.
The second purpose of the invention is to provide a method for quantitative detection of nucleic acid based on the ratiometric DNA molecular machine.
First, the present invention provides a ratiometric DNA molecular machine comprising the structure shown in fig. 1: the streptavidin magnetic beads are used as carriers, two sides of the streptavidin magnetic beads are respectively marked with biotin and sulfydryl anchor chains which are used as probes to complement with target molecules, and the gold nanoparticles are used as signal indicators; the target molecule is used as a walking molecule to trigger the DNA molecular machine, the endonuclease IV is used as fuel to drive the DNA molecular machine, and In is used as an internal standard element;
furthermore, the invention provides a method for quantitatively detecting nucleic acid based on the ratio type DNA molecular machine, which utilizes different amounts of DNA molecular machines triggered by target molecules with different concentrations and different amounts of released gold nanoparticles to measure the quantities of the gold nanoparticles197Au and internal standard element115And finally realizing accurate quantitative analysis on target molecules by the ICPMS signal ratio of In.
In the detection method, accurate quantitative analysis of target molecules is realized by combining a ratio type DNA molecular machine with an inorganic element mass spectrum for the first time. The principle is shown in FIG. 1, and the target molecule (walker) hybridizes to the anchor DNA on the magnetic beads through base pairing to form an enzyme recognition site. The site is cut by Endon IV enzyme, so that the anchor chain DNA is divided into two sections, wherein the AuNP marked DNA chain segment is separated from the DNA molecular machine orbit, and a walker and the next anchor chain DNA are hybridized to form a new enzyme recognition site to realize autonomous circular motion under dynamic balance. Internal standard element independent of target molecule after a large amount of AuNP marked DNA chain fragments released by molecular machine are digested115In was analyzed, elemental197Au and115the signal ratio of In and the concentration of the target molecule have a better linear relation In a certain range, so that the quantitative detection of the target molecule can be realized.
The detection method of the invention preferably comprises the following steps:
s1: synthesizing gold nanoparticles by a common sodium citrate reduction method;
s2: the anchor DNA was reacted with UDG enzyme at 37 ℃ for 1 hour;
s3: reacting the anchor chain DNA with a certain amount of thiol-modified T10Mixing the DNA and the gold nanoparticles, standing overnight, forming AU-S bonds by a salt raising method, and synthesizing anchor chains marked by the gold nanoparticles;
s4: reacting the anchor chain marked by the gold nanoparticles with magnetic beads marked by streptavidin overnight, and synthesizing a track of a DNA molecular machine;
s5: adding target molecules with the same volume and different concentrations to mix with the track, reacting at 37 ℃ for 1 hour to hybridize to form a DNA double chain, and magnetically separating and washing;
s6: adding endonuclease IV, identifying and cutting the base-lacking site, modifying the DNA segment with the gold nanoparticles to fall off from the track, hybridizing the target molecule with the anchor chain DNA on the next track, and identifying and cutting the next base-lacking site by the endonuclease IV to form an autonomous cyclic walking mode;
s7: magnetically separating the reacted solution, reacting with aqua regia for 10min, and coating with 1ng ml-1Diluted to 4mL.
S8: in solution for mass spectrometric recording of inorganic elements197Au and115signal ratio of In.
In the method of the invention, the anchor strand DNA described in S3 is reacted with thiol-modified T10The amount ratio of DNA used was 1: 5.
In the method of the invention, the salt raising method of S3 comprises the following steps: first, 50. mu.L of 10% (v/v) Tween 20 was added. The solution was then sonicated for 30 seconds immediately after the addition of a volume of 2M NaCl, and the mixed solution was then incubated at ambient temperature for 20 minutes. This process was repeated about 8 times until the NaCl concentration reached 0.8M. The solution after reaction was incubated overnight at room temperature, centrifuged and washed.
In the method of the present invention, the synthesis method of S4: the reaction conditions were shaken overnight at room temperature and the buffer for the reaction was 10mM Tris-HCl, 0.1% PEG 4000, 0.1% TW 20,50mM NaCl, pH 7.5.
In the method of the present invention, the amount of the orbital complex used in S5 is preferably 5. mu.L.
In the method, the reaction condition of S6 is that 0.1-0.5U endonuclease IV reacts for 0.5-4 hours at 25-65 ℃ by oscillation.
(III) advantageous effects
The invention has the beneficial effects that:
1. the DNA walker machine converts and amplifies nucleic acid signals in a sample into mass spectrum ratio signals through a combustion bridge mechanism, and the signals of the nucleic acid are highly amplified, so that accurate and quantitative analysis and determination of the nucleic acid with high sensitivity are realized.
2. The present invention enables magnetic separation of biosensors using Magnetic Microparticles (MMPs) after hybridization of DNA orbitals and target DNA, allowing the DNA machinery to subsequently function under optimal reaction conditions after matrix removal.
3. The invention provides a ratio method after adding internal standard elements. Unlike the use of monomers to determine the absolute value of ICPMS, this strategy measures target-dependent197Internal standard independent of Au isotope and target115The In isotope generates relative signals, and high stability and high accuracy of the signals are achieved.
The use of endo IV allows the detection of any DNA sequence by merely altering the sequence of the probe DNA, since only depurination/depyrimidination (AP) sites need to be designed in the probe DNA rather than specific recognition sequences. In combination with the specific recognition of aptamers and target molecules, the proposed DNA molecular machinery may be applicable to other targets, such as proteins and small biomolecules.
Drawings
FIG. 1 is a schematic diagram of a DNA molecular machine.
FIG. 2 is a graph showing the effect of the DNA orbital sequence of the present invention on the performance of a DNA molecular machine for detecting nucleic acids.
FIG. 3 is a graph showing the results of optimization of the optimum addition amount and the optimum reaction time of the Endon IV enzyme, which is a key substance of the present invention.
FIG. 4 is a graph showing the reaction temperature optimization results of the analysis method of the present invention.
FIG. 5 is a graph showing the results of the analysis specificity of the DNA detection by the analysis method of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The DNA molecular machine of the present invention comprises the following structure: carrier, probe, reporter, fuel.
Further, the carrier is streptavidin magnetic beads; the probe is a DNA sequence with two ends respectively labeled with biotin and sulfhydryl, and a partial sequence of the probe is complementary with a target molecule; the reporter is gold nanoparticles which are connected with the mercapto end of the probe; the fuel is endonuclease IV.
Further, the probe sequences are listed in Table 1.
Further, the endonuclease IV has a recognition site which is a base lacking site in the double-stranded DNA.
Further, the base deletion site of the probe sequence is generated by recognizing U base in the DNA sequence by UDG enzyme and removing.
The nucleic acid detection method comprises the following steps:
s1: mu.L of the above DNA molecular machine complex, 25. mu.L of water (blank) or 25. mu.L of the target DNA solution was added to a 200. mu.L PCR tube and mixed well.
S2: after incubating the above mixed solution at 37 ℃ for 1 hour, the complex was separated by a magnetic field and washed three times with 0.2mL of buffer.
S3: to the above 200. mu.L PCR tube, 1. mu.L EndoIV (200U/mL) solution, 10. mu.L reaction buffer and 89. mu.L water were added, mixed well and gently shaken at 37 ℃ for 2 hours.
S4, taking out the supernatant, adding 50 mu L of aqua regia to react for 10min.
S5 use 1ng mL-1In element solution the solution obtained above was diluted to 4mL and measured by ICP-MS197Au and115the intensity of the In element and the ratio thereof were calculated.
The embodiment of the invention comprises the following steps:
1. experimental apparatus and equipment used in embodiment of the invention
The instruments used in the experiment mainly include: MSC-100 constant temperature oscillator, NexION 350X inductively coupled plasma mass spectrometer.
2. Experimental reagent used in embodiment of the invention
All oligonucleotides were supplied by Shanghai Biotech, Inc. and the sequences are shown in Table 1.
Endonuclease iv (endo iv), uracil-DNA glycosylase (UDG enzyme), streptavidin-coated magnetic microspheres, chloroauric acid hydrate and tris- (hydroxymethyl) methylaminomethane, sodium chloride (NaCl), disodium hydrogen phosphate, sodium dihydrogen phosphate, tris (2-carboxyethyl) phosphine hydrochloride, tween 20 and sodium citrate.
Deionized Water (DIW, 18.2M. omega. cm) was obtained from Milli-Q Integral Water purification apparatus-1)。
Example 1:
s1, 5 different DNA molecular machine complexes are synthesized by using 5 different DNA strands.
S2: mu.L of the above 5 different DNA molecular machine complexes and 25. mu.L of the target DNA solution were added to a 200. mu.L LPCR tube and mixed well.
S3: after incubating the above mixed solution at 37 ℃ for 1 hour, the complex was separated by a magnetic field and washed three times with 0.2mL of buffer.
S4: to the above 200. mu.L PCR tube, 1. mu.L of Endo IV (200U/mL) solution, 10. mu.L of reaction buffer and 89. mu.L of water were added, mixed well and gently shaken at 37 ℃ for 2 hours.
S5, taking out the supernatant, adding 50 mu L of aqua regia to react for 10min.
S6 use 1ng mL-1In element solution the solution obtained above was diluted to 4mL and measured by ICP-MS197Au and115the intensity of the In element and the ratio thereof were calculated.
The experimental results are as follows:
in order to explore the influence of the DNA track sequence on the performance of the DNA molecular machine for detecting nucleic acid, the experiment designs 6 DNA track molecules, the structures of which are shown in figure 2a, and simultaneously carries out 6 groups of detection experiments under the premise of the same other reaction conditions and reagents, thereby exploring the relationship between the performance of the DNA molecular machine for detecting nucleic acid and the DNA track sequence. And performing blank tests respectively, wherein the blank tests are designed in such a way that the target DNA solution to be detected is replaced by pure water.
The results of the experiments are shown in FIG. 2b, A1, A2, A3, A4, A5 and A6 represent 6 different DNA molecular machines, and the 6 different designs are shown in FIG. 2 a. The U base will be recognized by the UDG enzyme to form an apurinic/Apyrimidinic (AP) site, which is the cleavage site for the endo IV enzyme.
S/N, which represents the ratio of the signal intensity to the blank intensity measured at the same concentration of the target DNA.
The dynamic equilibrium of the DNA walker is maintained by competition between the orthosteric hybridisation on the next anchor and the de-hybridisation of the remaining duplex on the previous anchor. The length of the 5' -upper arm of the anchor strand corresponds to the length of the foothold on the target strand, which will hybridize to the complementary segment of the neighboring anchor strand, inducing strand translocation from position n to position n + 1. This process is counteracted by a stronger hybridization of the target strand to the anchor strand at position n and the 3' -lower arm. As shown in FIG. 2b, the performance trend was observed as A5> A2> A1> A6> A4> A3, and it was hypothesized that when the hybridization energies of the two moieties were nearly equal, the opposite reaction between the two promoted the dynamic equilibrium and translocation performance of the DNA walker. To confirm the possibility of guesswork, we calculated each partial hybridization energy by NUPACK and the specific values are listed in table2. The calculation result is basically consistent with the experiment, and the experiment design can be guided by the rule. In addition to this, we observed that the 5 th molecular machine showed the best performance in these anchor chains (a1 to a 5). Therefore, we chose A5 and the corresponding target DNA (T5) as an example to study accurate and amplified DNA assays.
Example 2:
s1 Synthesis of the DNA molecular machine indicated A5.
S2: add 5. mu.L of the above DNA molecular machine complex and 25. mu.L of the target DNA solution to a 200. mu.L PCR tube and mix well.
S3: after incubating the above mixed solution at 37 ℃ for 1 hour, the complex was separated by a magnetic field and washed three times with 0.2mL of buffer.
S4: to the above 200. mu.L PCR tube was added a certain amount of EndoIV (200U/mL) solution, 10. mu.L reaction buffer and 89. mu.L water, mixed well and gently shaken at 37 ℃ for 2 hours.
S5, taking out the supernatant, adding 50 mu L of aqua regia to react for 10min.
S6 use 1ng mL-1In element solution the solution obtained above was diluted to 4mL and measured by ICP-MS197Au and115the intensity of the In element and the ratio thereof were calculated.
The experimental results are as follows:
the experiment is to optimize the addition amount and the reaction time of the Endon IV enzyme which is the key substance of the invention.
The DNA molecular machine in this experiment was designed as A5 (the specific sequence is as described in example 1), and the other reaction conditions were as described in example 2.
The concentrations of the Endon IV enzyme are respectively designed to be 0.1U, 0.2U, 0.4U and 0.5U. The reaction time is respectively designed to be 0.5h, 1h, 2h and 4 h.
As shown in fig. 3, the ordinate is IAu/IInThe values of (d) are, on the abscissa, the incubation time and the concentration of endo IV, respectively. Prolonged concentrations of endo IV and incubation times will increase both signal and background values, and to some extent will result in a decrease in S/N. Considering two factors of sensitivity and time consumption, we selected 0.2U and 2h, respectively, as the following experimental conditions.
Example 3:
s1 Synthesis of the DNA molecular machine indicated A5.
S2: add 5. mu.L of the above DNA molecular machine complex and 25. mu.L of the target DNA solution to a 200. mu.L PCR tube and mix well.
S3: after incubating the above mixed solution at 37 ℃ for 1 hour, the complex was separated by a magnetic field and washed three times with 0.2mL of buffer.
S4: to the above 200. mu.L PCR tube, 1. mu.L EndoIV (200U/mL) solution, 10. mu.L reaction buffer and 89. mu.L water were added, mixed well and gently shaken at a certain temperature for 2 hours.
S5, taking out the supernatant, adding 50 mu L of aqua regia to react for 10min.
S6 use 1ng mL-1In element solutionThe solution was diluted to 4mL and measured by ICP-MS197Au and115the intensity of the In element and the ratio thereof were calculated.
The experimental results are as follows:
this experiment is the optimum reaction temperature for the preferred invention.
In this experiment, the DNA molecular machine was designed to be A5 (the specific sequence is as described in example 1), the amount of Endon IV enzyme added and the reaction time were 0.2U and 2h, respectively (as described in example 2), and the other reaction conditions were as described in example 3.
The reaction temperatures were 25 deg.C, 37 deg.C, 45 deg.C, 55 deg.C, and 65 deg.C, respectively.
As shown in fig. 4, the effect of a range of temperatures on the performance of the present invention was studied to obtain an optimum temperature of 37 ℃. It may be difficult to separate the hybridized DNA fragments when the temperature is too low, further resulting in slow movement of the walker, while when the temperature is too high, the DNA walker is released resulting in reduced efficiency.
Example 4:
s1 Synthesis of the DNA molecular machine indicated A5.
S2: mu.L of the above DNA molecular machine complex and 25. mu.L of the same concentration of different DNA solution were added to a 200. mu.L PCR tube and mixed well.
S3: after incubating the above mixed solution at 37 ℃ for 1 hour, the complex was separated by a magnetic field and washed three times with 0.2mL of buffer.
S4: to the above 200. mu.L PCR tube, 1. mu.L EndoIV (200U/mL) solution, 10. mu.L reaction buffer and 89. mu.L water were added, mixed well and gently shaken at 37 ℃ for 2 hours.
S5, taking out the supernatant, adding 50 mu L of aqua regia to react for 10min.
S6 use 1ng mL-1In element solution the solution obtained above was diluted to 4mL and measured by ICP-MS197Au and115the intensity of the In element and the ratio thereof were calculated.
The experimental results are as follows:
this experiment was conducted to investigate the specificity of the DNA molecular machine of the present invention for detecting DNA molecules.
In this experiment, the DNA molecular machine was designed to be A5 (the specific sequence is as described in example 1), the amount of Endon IV enzyme added and the reaction time were 0.2U and 2h, respectively (as described in example 2), the reaction temperature was 37 deg.C (as described in example 3), and the other reaction conditions were as described in example 4.
The four different sequences are target DNA, single base mismatch, double base mismatch and random sequence, and the specific sequences are shown in Table 1.
After the reaction is finished, detecting by using ICPMS197Au and115the intensity of the In element and its ratio are calculated, and the image is drawn according to the corresponding ratios obtained from the different sequences, as shown In fig. 5. The results show the I of the ratiometric DNA molecular machine for different target sequences under the same conditionsAu/IInAnd (6) responding. We observed that the ratio of single base mismatches, double base mismatches and random sequences can be clearly distinguished from the ratio corresponding to the perfectly matched target DNA, indicating that the ratiometric DNA molecular machinery is able to distinguish between single nucleotide differences with high specificity.
Table 1.DNA sequences used in this work.
Figure BDA0002130958120000081
Letters in bold represent the location and type of DNA modifications
U：/ide oxy u/
Table2. hybridization energy and ratio of the two parts.
Figure BDA0002130958120000082

Claims (9)

1. A ratiometric DNA molecular machine, useful for biological analysis.
2. The ratiometric DNA molecular machine of claim 1, wherein: the ratio type DNA molecular machine is a DNA walker which consists of three parts, namely a magnetic bead, an anchor chain and gold nanoparticles, wherein the magnetic bead is used as a carrier, the anchor chain is used as a probe to be complementary with a target molecule, and the gold nanoparticles are used as a reporter.
3. The ratiometric DNA molecular machine of claim 1, wherein: the ratio type DNA molecular machine is triggered by target molecules, endonuclease IV is used as a driving force, and walking of the target molecules is realized through a 'bridge combustion' mechanism so as to convert and amplify biological signals into mass spectrum ratio signals.
4. The ratiometric DNA molecular machine of claim 1, wherein: the specific recognition site of the endonuclease IV is a base lacking site of double-stranded DNA.
5. The ratiometric DNA molecular machine of claim 1, wherein: the anchor DNA sequence contains a portion complementary to the target molecule sequence.
6. The ratiometric DNA molecular machine of claim 1, wherein: the anchor DNA having U bases is reacted with UDG enzyme to remove U bases and form an abasic-containing anchor.
7. The ratiometric DNA molecular machine is used for nucleic acid (target molecule) analysis, characterized by: comprises the following steps
Reacting the anchor strand DNA of claim 1 with a UDG enzyme;
synthesizing a trajectory of the ratiometric DNA molecular machine of claim 1;
adding target molecules with the same volume and different concentrations to mix with the track, reacting and hybridizing at a certain temperature to form a DNA double chain, and magnetically separating and washing;
adding endonuclease IV, identifying and cutting the base-lacking site, modifying the DNA segment with the gold nanoparticles to fall off from the track, hybridizing the target molecule with the anchor chain DNA on the next track, and identifying and cutting the next base-lacking site by the endonuclease IV to form an autonomous cyclic walking mode;
magnetically separating the reacted solution, reacting with aqua regia, and coating with 1ng ml-1Diluted to volume.
In solution for mass spectrometric recording of inorganic elements197Au and 115the signal intensity of In and the ratio thereof was calculated.
8. The method for analyzing nucleic acid according to claim 7, wherein: in step S2, the synthesis mechanism is the binding of biotin to streptavidin and the formation of AU-S bonds.
9. The method for analyzing nucleic acid according to claim 7, wherein: in step S4, 0.1-0.5U of endonuclease IV is added, and the mixture is subjected to an oscillating reaction at 25-65 ℃ for 0.5-4 hours.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160312277A1 (en) * 2008-01-14 2016-10-27 Applied Biosystem, Llc Amplification and detection of ribonucleic acids
US20170168044A1 (en) * 2014-06-09 2017-06-15 Xiamen University Quantitative analysis method based on air pressure measuring
CN107202785A (en) * 2017-05-24 2017-09-26 湖南大学 The method that sketch-based user interface type SERS sensing principles detect heavy metal ion
CN107462704A (en) * 2017-09-21 2017-12-12 清华大学深圳研究生院 A kind of biology sensor and preparation method thereof, concentration of target molecules detection method
CN109752362A (en) * 2019-01-10 2019-05-14 济南大学 A kind of biosensor and preparation method thereof detecting uracil-DNA glycosylase

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160312277A1 (en) * 2008-01-14 2016-10-27 Applied Biosystem, Llc Amplification and detection of ribonucleic acids
US20170168044A1 (en) * 2014-06-09 2017-06-15 Xiamen University Quantitative analysis method based on air pressure measuring
CN107202785A (en) * 2017-05-24 2017-09-26 湖南大学 The method that sketch-based user interface type SERS sensing principles detect heavy metal ion
CN107462704A (en) * 2017-09-21 2017-12-12 清华大学深圳研究生院 A kind of biology sensor and preparation method thereof, concentration of target molecules detection method
CN109752362A (en) * 2019-01-10 2019-05-14 济南大学 A kind of biosensor and preparation method thereof detecting uracil-DNA glycosylase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG CHAOQUN等: "Ratiometric DNA Walking Machine for Accurate and Amplified Bioassay", 《CHEM EUR J》 *

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