AU2020101877A4 - DNA hydrogel based on signal amplification of biomimetic enzymes and application thereof - Google Patents
DNA hydrogel based on signal amplification of biomimetic enzymes and application thereof Download PDFInfo
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Abstract
The invention relates to a DNA hydrogel based on signal amplification of biomimetic
enzymes and application thereof to the detection of microcystin-LR, where a DNA
hydrogel coating layer is used to coat the biomimetic enzymes having a peroxidase
activity, and an aptamer of the microcystin-LR is used as a cross-linking bridge when the
hydrogel coating layer is constructed. Hereby, when the DNA hydrogel encounters the
microcystin-LR, the structure of the cross-linking bridge in the structure of the DNA
hydrogel changes, which disintegrates the DNA hydrogel to release the biomimetic
enzymes; and the biomimetic enzymes can catalyze a chromogenic reaction, and then the
concentration or content of the microcystin-LR can be detected in combination with a
colorimetric detection means. Compared with the traditional high-performance liquid
chromatography (HPLC), enzyme-linked immunosorbent assay, liquid
chromatography-tandem mass spectrometry and other detection methods, the invention
has the advantages such as low cost, high detection speed, and good portability of
supporting detection devices.
1/2
* 0 .*00
a 0 CL 10
0 a ' 0
0 V 0 H'02~
1 P-SA 4{ >P-SB /SAptamner 0 Cu/Au/Pt NPs *MC-LR TMB 'Aptamer-MC-LR complex
FIG. 1
12
8
4
-4
-8 2-Aptamer+SA+SB
3-Aptamer
-12 -4-Aptamer+SA+SB+MC-LR
220 240 260 280 300 320
Wavelength (nm)
FIG. 2
Description
1/2
* 0 .*00 a 0CL10 0 a ' 0
0 V 0 H'02~
1 P-SA 4{ >P-SB /SAptamner 0 Cu/Au/Pt NPs *MC-LR TMB 'Aptamer-MC-LR complex
FIG. 1
12
8
4
-4
-8 2-Aptamer+SA+SB 3-Aptamer -12 -4-Aptamer+SA+SB+MC-LR
220 240 260 280 300 320 Wavelength (nm) FIG. 2
[0001] Technical Field
[0002] The invention belongs to the technical field of water environment
monitoring, and in particular, relates to a DNA hydrogel based on signal amplification of
biomimetic enzymes and application thereof to the detection of microcystin-LR.
[0003] Description of Related Art
[0004] Due to water eutrophication, cyanobacteria blooms have occurred more and
more frequently in rivers and lakes across the world. Microcystins (MCs) produced and
released by cyanobacteria such as Anabaena, Aphanizomenon, Clindrospermopsis and
Nodularia have aroused widespread attentions. The MC is a kind of cyclic heptapeptide
compound having a biological activity, and can exist stably in the environment. Among
them, microcystin-LR (MC-LR for short) is the most abundant (approximately accounting
for 99.8% of the total concentration of the MCs in natural water) and the most toxic,
which has seriously threatened the ecological safety of water bodies and human health. A
large number of studies have shown that even in the case of long-term low-level exposure,
the MC-LR exposure is highly correlated with the occurrence of liver cancer. The MC-LR
may inhibit the activity of protein phosphatase and affect the regulation of miRNA
expression, damaging the kidneys, skin and reproductive system. Furthermore, recent
studies have found that the MC-LR is also neurotoxic, and the exposure to the MC-LR
may cause blood-brain barrier disruption, memory deficits and Alzheimer's disease-like
symptoms. MC-LR contamination has been reported around the world. For example, in
1996, 116 patients in Brazil got poisoned due to the use of MC-contaminated water during
hemodialysis treatment, resulting in 52 deaths; and in the south of Yellow River Basin in
China, water blooms have frequently occurred to about 70% of lakes and ponds, of which
about 80% contain MCs, and the MCs have been detected in important large freshwater
lakes such as Taihu Lake, Chaohu Lake and Dianchi Lake. The World Health Organization
(WHO) recommends that the limit of MC-LR in drinking water is 1.0 [g/L. Furthermore,
the MC-LR may also be enriched in aquatic products through the action of a food chain,
bringing potential hazards to food safety. Therefore, it is of important significance and
necessity in the fields of environment safety, food safety and human health to develop a
simple, sensitive, rapid, and reliable analysis method to enable the trace monitoring of the
[0005] (I) Technical Problems to be Solved
[0006] In order to solve the above-mentioned problems in the prior art, the
invention provides a DNA hydrogel based on signal amplification of biomimetic enzymes
and application thereof to the detection of microcystin-LR, where the DNA hydrogel is
used to coat the biomimetic enzymes. When the DNA hydrogel encounters the
microcystin-LR, the structure of a cross-linking bridge in the structure of the DNA
hydrogel changes, which disintegrates the DNA hydrogel to release the biomimetic
enzymes; and the biomimetic enzymes can catalyze a chromogenic reaction, and then the
concentration or content of the microcystin-LR can be detected in combination with a
colorimetric detection means. Compared with the traditional high-performance liquid
chromatography (HPLC), enzyme-linked immunosorbent assay, liquid
chromatography-tandem mass spectrometry and other detection methods, the invention
has the advantages such as low cost, high detection speed, and good portability of
supporting instruments.
[0007] (II) Technical Solutions
[0008] To achieve the objective above, the technical solutions employed by the
invention mainly include the following ones.
[0009] In one aspect, the invention provides a DNA hydrogel based on signal
amplification of biomimetic enzymes, and the DNA hydrogel comprises: a hydrogel
coating layer and biomimetic enzymes coated with the hydrogel coating layer;
[0010] the biomimetic enzymes are biomimetic enzymes that are compounded
from precious metals or transition metals and have a peroxidase activity;
[0011] the hydrogel coating layer comprises a straight-chain polymer backbone, a
first short-chain DNA, a second short-chain DNA and an MC-LR aptamer, and the first
short-chain DNA and the second short-chain DNA are respectively designed according to
a base sequence of the MC-LR aptamer and according to the principle of base
complementary pairing, so that the first short-chain DNA and the second short-chain DNA
may be complementarily paired with part of bases of the MC-LR aptamer,
[0012] wherein the first short-chain DNA is grafted on the straight-chain polymer
backbone to form a first DNA-containing backbone, and the second short-chain DNA is
grafted on the straight-chain polymer backbone to form a second DNA-containing
backbone; and the first DNA-containing backbone and the second DNA-containing
backbone are cross-linked by means of the MC-LR aptamer as a cross-linking bridge, to
form the hydrogel coating layer.
[0013] According to a preferred example of the invention, the biomimetic enzymes
are Au/Pt TNPs (peroxidases) formed by compounding Au and Pt, or the biomimetic
enzymes are Cu/Au/Pt TNPs (peroxidases) formed by compounding Cu with Au and Pt.
[0014] These biomimetic enzymes have the enzyme activities similar to those of
the natural enzymes, and the biomimetic enzymes not only have the peroxidase-like
activity, but also have the advantages such as good stability, reliable catalytic performance less susceptible to the environment, and low economic benefits. They can catalyze H202 to allow TMB (tetramethylbenzidine) to undergo a reaction with a color change.
[0015] Furthermore, compared with the single metal material, the complex
biomimetic enzymes (Cu/Au/Pt TNPs) that are synthesized from precious metals (Au, Pt)
or a transition metal (Cu) are significantly enhanced in the enzyme activity of the
peroxidases (TNPs), with high stability and high cost-effectiveness.
[0016] According to a preferred example of the invention, the straight-chain
polymer backbone is a straight-chain polyacrylamide backbone, a straight-chain
carboxymethyl cellulose backbone, a straight-chain N-isopropylacrylamide backbone or a
straight-chain polyvinyl alcohol backbone. More preferably, the straight-chain polymer
backbone is a straight-chain polyacrylamide backbone, which contains rich reactive amino
groups that are liable to reacting with the short-chain DNA containing bases to graft the
short-chain DNA to the linear backbone thereof.
[0017] According to a preferred example of the invention, a sequence of the
MC-LR aptamer is: 5'-GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC
CAC CTC ATT ATG CCC CAT CTC CGC-3'; a high-affinity region of the sequence of
the MC-LR aptamer is selected to design the first short-chain DNA and the second
short-chain DNA that are partially complementary to the aptamer; and a sequence of the
high-affinity region of the MC-LR aptamer is: 5'-A
TACCACCTCATTATGCCCCATCTCCGC-3'.
[0018] According to a preferred example of the invention, a sequence of the first
short-chain DNA is: 5'-Acrydite-TTT TTT GGG CAT AAT GAG-3'; and a sequence of
the second short-chain DNA is: 5'-Acrydite-TTT TTT GGG CAT AAT GAG-3'.
[0019] The above-mentioned first and second short-chain DNAs are characterized
in that they can stably hybridize with the MC-LR aptamer, and can ensure that when the
MC-LR aptamer encounters the MC-LR, the MC-LR can substitute the first and second short-chain DNAs in the MC-LR aptamer through competition. In other words, the binding force of the MC-LR to the aptamer is superior to that of the first and second short-chain DNAs; and only in this way can the MC-LR destroy the cross-linking structure of the DNA hydrogel, thereby disintegrating the DNA hydrogel to release the biomimetic enzymes TNPs therein.
In another aspect, the invention also provides a reagent composition for detecting
microcystin-LR, and the reagent composition comprises: the DNA hydrogel based on the
signal amplification of the biomimetic enzymes according to any one of the
above-mentioned solutions, a peroxide oxidizer and a reductive chromogenic agent.
[0020] Preferably, the peroxide oxidizer is H202 and the reductive chromogenic
agent is tetramethylbenzidine TMB. Preferably, the reagent composition further comprises
a buffer solution and concentrated hydrochloric acid.
[0021] In a further aspect, the invention provides a method for detecting
microcystin-LR, and the method includes the following steps:
[0022] Step 1: adding a certain amount of the above-mentioned DNA hydrogel
based on the signal amplification of the biomimetic enzymes to a sample solution to be
detected, and incubating the sample solution at 30-40°C for 0.5-2 h;
[0023] Step 2: adding hydrogen peroxide H202 and the chromogenic agent TMB
to a mixing system, adjusting pH with a buffer solution to weak acidity, reacting at the
room temperature, and then adding concentrated hydrochloric acid to terminate the
reaction; and
[0024] Step 3: performing quantitative determination using a colorimetric
detection method.
[0025] Among them, in Step 3, an absorbance value at a specific wavelength is
measured using an ultraviolet-visible spectrophotometer; and a standard curve is drawn
according to a series of standard solutions with known concentrations for quantitative determination.
[0026] Furthermore, the invention also provides a preparation method of a DNA
hydrogel based on signal amplification of biomimetic enzymes, and hte method comprises
the following steps:
[0027] Si: preparing biomimetic enzymes Cu/Au/Pt TNPs having a peroxidase
activity:
[0028] mixing a soluble copper salt and a complexing agent in water; then adding
a sufficient amount of a reducing agent; stirring for reaction, and then adding an
appropriate amount of an auric acid or aurate and a platinic acid or platinate; stirring again
for reaction to prepare the biomimetic enzymes Cu/Au/Pt TNPs having the peroxidase
activity;
[0029] S2: preparing an aptamer as well as a first short-chain DNA and a second
short-chain DNA,
[0030] wherein a sequence of an MC-LR aptamer is: 5'-GGC GCC AAA CAG
GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3';
a sequence of afirst short-chain DNA is: 5'-Acrydite-TTT TTT GGG CAT AAT GAG-3';
and a sequence of a second short-chain DNA is: 5'-Acrydite-TTT TTT GTA ATT GTC
ATG-3', and
[0031] S3: preparing the DNA hydrogel based on the signal amplification of the
biomimetic enzymes, comprising the following sub-steps:
[0032] S31: dissolving the first short-chain DNA and the second short-chain DNA
in a buffer solution respectively to obtain a solution of the first short-chain DNA and a
solution of the second short-chain DNA;
[0033] S32: preparing a solution of a polymer monomer which can be polymerized
to generate a straight-chain polymer for DNA grating;
[0034] blending the solution of the first short-chain DNA with the solution of the polymer monomer, and adding a polymerization initiator and a catalyst in a vacuum environment to obtain a straight-chain polymer grafted with the first short-chain DNA;
[0035] blending the solution of the second short-chain DNA with the solution of
the polymer monomer, and adding a polymerization initiator and a catalyst in a vacuum
environment to obtain a straight-chain polymer grafted with the second short-chain DNA;
and
[0036] S33: mixing the straight-chain polymer grafted with the first short-chain
DNA and the straight-chain polymer grafted with the second short-chain DNA, adding the
biomimetic enzymes Cu/Au/Pt TNPs prepared in Step Si, then adding an aptamer solution
acting as a cross-linking bridge, fully mixing for reaction, and cooling to the room
temperature to obtain the DNA hydrogel based on the signal amplification of the
biomimetic enzyme.
[0037] Preferably, in Step Si, the auric acid is a chloroauric acid; the aurate is
chloroaurate; the platinum acid is tetrachloroplatinic acid or hexachloroplatinic acid; and
the platinate is tetrachloroplatinate or hexachloroplatinate.
[0038] Preferably, in Sub-step S32, the solution of the polymer monomer is an
acrylamide solution, the polymerization initiator is an ammonium persulfate solution, and
the catalyst is an N,N,N,N-tetramethylethylenediamine (TEMED) solution.
[0039] Preferably, in Step S33, the straight-chain polymer grafted with the first
short-chain DNA and the straight-chain polymer grafted with the second short-chain DNA
are mixed at a molar ratio of 1:1; after mixing, a PBS buffer solution is added; then an
aptamer solution as a crosslinking bridge is added, mixed vigorously for reacting at 65°C,
and cooled to the room temperature to form a hydrogel.
[0040] (III) Beneficial Effect
[0041] The invention has the following beneficial effects.
[0042] (1) The invention provides the DNA hydrogel based on the signal amplification of the biomimetic enzymes, where the DNA hydrogel is used to coat the biomimetic enzymes. When the DNA hydrogel encounters the microcystin-LR, the binding of the cross-linking bridge (the aptamer) in the structure of the DNA hydrogel allows a change in a secondary structure of the aptamer, thereby destroying the cross linking of the aptamer to disintegrate the DNA hydrogel for releasing the biomimetic enzyme; and the biomimetic enzymes can catalyze a chromogenic reaction to present a visible change in color. The color depth is directly correlated to the amount of the MC-LR, therefore, the visual detection of the MC-LR can be realized according to the final color of the solution. For example, the concentration or content of the microcystin-LR can be measured in combination with a colorimetric detection means. Compared with the traditional high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay, liquid chromatography-tandem mass spectrometry and other detection methods, the invention has the advantages such as low cost, high detection speed, and cheap and portable detection instruments. Among them, the colorimetric detection means may be a colorimetric sensor or an ultraviolet-visible spectrophotometer which can quantitatively detect the MC-LR.
[0043] (2) The DNA hydrogel based on the signal amplification of the biomimetic
enzymes according to invention contains the embedded biomimetic enzyme having the
natural enzyme activity, and the biomimetic enzyme not only has the peroxidase-like
activity, but also has the advantages such as good stability, reliable catalytic performance
less susceptible to the environment, and low economic benefits. The biomimetic enzymes
are complex biomimetic enzymes (Cu/Au/Pt TNPs) that are synthesized from precious
metals (Au, Pt) or a transition metal (Cu). Compared with the single metal material, the
enzyme activity of the peroxidases (TNPs) of the precious metals is significantly enhanced,
with high stability and high cost-effectiveness, and can be produced and applied on a large
scale.
[0044] According to the invention, by means of the fact that the DNA hydrogel
undergoes a gel-sol transition or a signal-controlled rigid change when triggered by a
signal, the biomimetic enzymes having the peroxidase (TNPs) activity are encapsulated in
the DNA hydrogel, and the state of the gel is changed by virtue of the responses of the
DNA hydrogel against target molecules to specifically release the embedded enzymes for
outputting visual signals. Among them, the use of the biomimetic enzymes makes signal
amplification more stably, and thus can be used to detect trace targets; and the hydrogel
coating layer not only ensures that the signal output will specifically vary with the change
in the concentration/amount of a target, but also can provide a more stable environment
for the biomimetic enzymes to favorably allow the biomimetic enzymes to maintain the
catalytic activity for a long time.
[0045] The technical solutions of the embodiments of the invention will be
understood more clearly below by making a brief illustration to the drawings to be used in
the embodiments. It is evident that the drawings described in the following only relate to
some embodiments of the invention. Other drawings may be obtained according to these
drawings by those ordinarily skilled in the art without creative efforts.
[0046] FIG. 1 is a schematic design diagram of a detection method according to
the invention.
[0047] FIG. 2 shows the design and competitiveness verification of
complementary short chains.
[0048] FIG. 3 shows responses of a Cu/Au/Pt-DNA hydrogel to different
concentrations of MC-LR.
[0049] The invention will be described in detail through specific embodiments
below in conjunction with the accompanying drawings for a better explanation and
understanding of the invention.
[0050] The invention provides a DNA hydrogel based on signal amplification of
biomimetic enzymes, and the DNA hydrogel comprises: a hydrogel coating layer and
biomimetic enzymes coated with the hydrogel coating layer;
[0051] the biomimetic enzymes are biomimetic enzymes that are compounded
from precious metals or transition metals and have a peroxidase activity;
[0052] the hydrogel coating layer comprises a straight-chain polymer backbone, a
first short-chain DNA, a second short-chain DNA and an MC-LR aptamer, and the first
short-chain DNA and the second short-chain DNA are respectively designed according to
a base sequence of the MC-LR aptamer and according to the principle of base
complementary pairing, so that the first short-chain DNA and the second short-chain DNA
may be complementarily paired with part of bases of the MC-LR aptamer,
[0053] wherein the first short-chain DNA is grafted on the straight-chain polymer
backbone to form a first DNA-containing backbone, and the second short-chain DNA is
grafted on the straight-chain polymer backbone to form a second DNA-containing
backbone; and the first DNA-containing backbone and the second DNA-containing
backbone are cross-linked by means of the MC-LR aptamer as a cross-linking bridge, to
form the hydrogel coating layer.
[0054] Among them, the biomimetic enzymes are Au/Pt that are formed by
compounding Au and Pt and have a peroxidase activity, or Cu/Au/Pt that are formed by
compounding Cu with Au and Pt and have a peroxidase activity. The use of the
biomimetic enzymes having the peroxidase activity to replace the natural TNPs can
increase enzyme stability and catalytic environmental tolerance, and reduce costs. The biomimetic enzymes TNPs can catalyze H202 to allow TMB (tetramethylbenzidine) to undergo a reaction with a color change.
[0055] Among them, the straight-chain polymer backbone is a straight-chain
polyacrylamide backbone, a straight-chain carboxymethyl cellulose backbone, a
straight-chain N-isopropylacrylamide backbone, a straight-chain polyvinyl alcohol
backbone, or the like, as long as these straight-chain/linear polymers can be easily grafted
by the short-chain DNA.
[0056] Among them, the sequence of the MC-LR aptamer is: 5'-GGC GCC AAA
CGC-3'. When the first and second short-chain DNAs are designed, it is necessary to
ensure that the designed first and second short-chain DNAs can stably hybridize with the
aptamer, and have a binding capability weaker than the binding capability of the MC-LR
to the aptamer, so that when the prepared DNA hydrogel encounters the MC-LR, the
MC-LR can bind to the aptamer to disconnect the aptamer from the first and second
short-chain DNAs, leading to structural collapse of the hydrogel and release of the
embedded biomimetic enzymes TNPs.
[0057] As shown in FIG. 1, a schematic diagram of the application of the DNA
hydrogel based on the signal amplification of the biomimetic enzymes as developed by the
invention to the MC-LR detection.
[0058] The aptamer of the MC-LR is taken as a cross-linking bridge of the DNA
hydrogel, and two short-chain DNAs (SA, SB) that are partially complementary to the
aptamer are designed according to the principle of complementary base pairing. The two
short-chain DNAs (SA, SB) are grafted onto the straight-chain polyacrylamide backbone
respectively to form P-SA and P-SB. When the aptamer is added, both the P-SA and the
P-SB hybridize with the aptamer respectively to form a DNA-containing hydrogel coating
layer, which can coat the biomimetic enzymes Cu/Au/Pt having the peroxidase activity therein. When the hydrogel of such a structure system encounters the MC-LR, the MC-LR binds to the aptamer (to substitute the aptamer from the short-chain DNAs SA and SB through competition) to change the secondary structure of the aptamer, thereby destroying the hybridization between the aptamer and the short-chain DNAs (SA, SB), destroying the cross-linking structure, collapsing the structure of the hydrogel (the MC-LR that has bound to the aptamer loses the destructive capability), and releasing the embedded biomimetic enzymes Cu/Au/Pt TNPs. Subsequently, the Cu/Au/Pt TNPs catalyze H202 to oxidize TMB, which has a visible color change after being oxidized and presents a final color directly correlated to the amount of the MC-LR. Namely, the more the MC-LR, the more the structures of the hydrogel are collapsed; and the more the collapsed structures of the hydrogel, the more the biomimetic enzymes are released. Therefore, the visual detection of the MC-LR is enabled according to the final color of the solution. For example, the MC-LR can be quantitatively detected using an ultraviolet-visible spectrophotometer.
[0059] The description is made in detail below in conjunction with the particular
embodiments of the invention.
[0060] Example 1
[0061] In this example, a DNA hydrogel based on signal amplification of
biomimetic enzymes was prepared, wherein the biomimetic enzymes were Cu/Au/Pt with
peroxidase activity, and a straight-chain polymer backbone used in an outer hydrogel
coating layer was a straight-chain polyacrylamide backbone. The following provides a
preparation method of the DNA hydrogel according to this example:
[0062] Step 1: Preparation method of Cu/Au/Pt TNPs.
[0063] The biomimetic enzymes Cu/Au/Pt TNPs having a peroxidase activity were
synthesized with a "one-pot" method. 12 L of CuSO4 . 5H20 (0.1mol/L) and 25 L of
trisodium citrate (0.1 mol/L) were added to 10 mL of ultrapure water and then mixed evenly; then, 500 L of KBH4 (25 mmol/L) was added and stirred for 15 min; and 25 L of HAuCl4 (0.1mol/L) and 25 L of K2PtCl4 (0.1mol/L) were dropped into a resulting mixture solution respectively and stirred for 20 min to obtain the Cu/Au/Pt TNPs.
[0064] Step 2: Design of first and second short-chain DNAs.
[0065] The design principle includes ( and @ as follows.
[0066] 0 The first short-chain DNA and the second short-chain DNA were
respectively designed according to a base sequence of the MC-LR aptamer and according
to the principle of base complementary pairing, so that the first short-chain DNA and the
second short-chain DNA may be complementarily paired with part of bases of the MC-LR
aptamer.
[0067] @The designed first short-chain DNA (named as SA) and the designed
second short-chain DNA (named as SB) should stably hybridize with the aptamer and
meet the requirement that the MC-LR can successfully substitute the SA and SB from the
aptamer through competition (refer to the subsequent verification regarding this).
[0068] According to the above principles, on the NUPACK website, the
short-chain DNAs (SA, SB) with the following sequence were finally designed according
to the base sequence (5'-A TACCACCTCATTATGCCCCATCTCCGC-3') of a high
affinity region of the MC-LR aptamer:
SA 5'-Acrydite-TTT TTT GGG CAT AAT GAG-3' SB 5'-Acrydite-TTT TTT GTAATT GTC ATG-3' Aptamer 5'-GGC GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3'
[0069] Step 3: Preparation of the DNA hydrogel based on the signal amplification
of the biomimetic enzymes.
[0070] The preparation method of the Cu/Au/Pt-DNA hydrogel includes two steps:
[0071] (1) The first short-chain DNA (SA) and the second short-chain DNA (SB) were dissolved in a PBS (pH=7.4) buffer solution respectively to prepare a stock solution with a final concentration of 300 [mol/L.
[0072] 25% of an acrylamide solution, 10% of an ammonium persulfate (APS) and
% of an N,N,N,N-tetramethylethylenediamine (TEMED) solution were prepared
respectively. Among them, the ammonium persulfate was a polymerization initiator, and
the TEMED was a polymerization catalyst.
[0073] 5 pL of SA (300 mol/L) and 2.4 L of acrylamide (25%) were mixed
vigorously and then react in vacuum for 10 min in a vacuum drying oven; and 2.1 L of
the ammonium persulfate (10%), 4.2 L of the TEMED (5%) and 1.3 L of the PBS
(pH=7.4) were added, mixed vigorously and then reacted in vacuum for 15 min to obtain a
linear (straight-chain) polymer polymerization product P-SA grafted with the SA. A linear
(straight-chain) polymer polymerization product P-SB grafted with the SB was prepared
with the same method.
[0074] (2) The P-SA and the P-SB were mixed at a ratio of 1:1; the PBS (pH=7.4)
buffer solution was added; then, 4 L of Cu/Au/Pt TNPs was added; then, an aptamer
solution (2 L 35 mol/L) as a cross-linking bridge was added; a resulting mixture was
mixed vigorously for reacting at 65 °C for 5 min; the process of mixing vigorously for
reacting at 65 °C for 5 min was repeated; a resulting product was cooled to room
temperature to form a hydrogel, which was the DNA hydrogel based on the signal
simplification of the biomimetic enzymes.
[0075] Example 2
[0076] In this example, whether the binding competitiveness of the first
short-chain DNA (SA) and the second short-chain DNA (SB), as designed in Example 1,
against the aptamer meets the points (- @ in the design principles were verified. A
verification method was as follows:
[0077] (1) Sample preparation:
No. SA SB Apt PBS MC-LR (100 pmol/L) (100 mol/L) (100 mol/L) (10 ng/mL) Apt (aptamer) 0 0 18 L 182 L 0 MC-LR 0 0 0 182 L 18 L (microcystin-LR) Apt+cDNA 18 L 18 L 18 L 146 L 0 (aptamer+short-chai n DNA) Apt+cDNA+MC-LR 18 L 18 L 18 L 128 L 18 L (aptamer+short-chai n DNA+microcystin)
[0078] Note: Before the MC-LR was added, the mixture was denatured at 95°C for
minutes, cooled to room temperature and placed at 4°C for 20 minutes; and after the
MC-LR was added, the mixture was incubated at 37°C for 40 minutes and stored in a
refrigerator at -20°C as a sample to be detected.
[0079] (2) 200 L of the samples in the above table were placed in a cuvette
respectively, and then measured on a circular dichroism instrument. The parameters of the
circular dichroism instrument were set as follows: measuring wavelength range: 200-350
nm; scanning speed: 100 nm/min; response time: 4s; bandwidth: 2.0 nm; and scanning
mode: continuous. Baseline correction: a PBS solution.
[0080] (3) Data analysis.
[0081] The obtained original circular dichroism data were imported into origin for
plotting, with the results shown in FIG. 2.
[0082] As shown in FIG. 2, its ordinate is the circular dichroism. As can be known
from FIG. 2,
[0083] () The Apt of the MC-LR has obvious positive and negative peak shapes
at 280 nm and 245 nm, and a quadrature axis with the baseline is at about 260 nm, which
is a standard B-type DNA.
[0084] @When the SA and SB complementary to the Apt are added, the positive
peak of the Apt at 280 nm is red-shifted, and the negative peak at 245 nm is blue-shifted,
which is basically a B-type DNA, but with obviously reduced peak shape. This indicates
that the Apt binds to the SA and SB and changes the secondary structure of the Apt.
[0085] @ When the MC-LR is added to the composite system of Apt+SA+SB, a
resulting peak shape is obviously higher than that of Apt+SA+SB. The reason is that the
binding between the MC-LR and the Apt makes the complementary chains of the SA and
SB that are originally hybridized on the Apt fall off, the Apt returns to its original
configuration, and meanwhile, due to the existence of free SA and SB, its peak shape is
higher than that of the Apt.
[0086] It thus shows that the short-chain DNAs (SA, SB) involved meet the
established design requirements.
[0087] Example 3
[0088] In this example, the DNA hydrogel based on the signal amplification of the
biomimetic enzymes as prepared in Example 1 was applied to the quantitative detection of
the MC-LR in water. The detection method is as follows.
[0089] 10 pL of the prepared Cu/Au/Pt-DNA hydrogel from Example 1 was added
to a 1.5 mL disposable centrifuge tube, and a sample solution (containing the MC-LR) to
be tested was added and incubated at 37 C for 40 min. 198 L of H20(pH=3), TMB with
a final concentration of 26.64 mmol/L and H202with a final concentration of 48 mmol/L
were added to a mixed system for reaction at the room temperature, and concentrated
hydrochloric acid was added to terminate the reaction. An absorbance value at a specific
wavelength (optimally at 452 nm) was measured using an ultraviolet-visible
spectrophotometer.
[0090] According to a series of standard solutions with known MC-LR
concentrations, the absorbance value at the wavelength of 452 nm was measured
according to the foregoing method, and a concentration-absorbance standard curve was
drawn for quantitative determination.
[0091] As shown in FIG. 3, the sample solutions with the concentration of 0.00
[tg/L, 0.04 g/L, 0.40 g/L and 4.00 g/L were selected and tested in absorbance curve at
the wavelength of 350 nm-550 nm according to the above-mentioned method, and the
wavelength for the maximum absorbance was determined to be 452 nm. The quantitative
measurement technology of the spectrophotometer is a well-known conventional
technology at present, and will not be repeated here.
[0092] The description above only provides preferred embodiments of the present
invention. It should be noted that for those of ordinary skills in the art, various
improvements and modifications that can be made without departing from the principle of
the present invention shall be construed as falling within the protection scope of the
present invention.
Claims (12)
1. A DNA hydrogel based on signal amplification of biomimetic enzymes,
characterized in that, the DNA hydrogel comprises: a hydrogel coating layer and
biomimetic enzymes coated with the hydrogel coating layer;
the biomimetic enzymes are biomimetic enzymes that are compounded from
precious metals or transition metals and have a peroxidase activity;
the hydrogel coating layer comprises a straight-chain polymer backbone, a first
short-chain DNA, a second short-chain DNA and an MC-LR aptamer, and the first
short-chain DNA and the second short-chain DNA are respectively designed according to
a base sequence of the MC-LR aptamer and according to the principle of base
complementary pairing, so that the first short-chain DNA and the second short-chain DNA
may be complementarily paired with part of bases of the MC-LR aptamer,
wherein the first short-chain DNA is grafted on the straight-chain polymer
backbone to form a first DNA-containing backbone, and the second short-chain DNA is
grafted on the straight-chain polymer backbone to form a second DNA-containing
backbone; and the first DNA-containing backbone and the second DNA-containing
backbone are cross-linked by means of the MC-LR aptamer as a cross-linking bridge, to
form the hydrogel coating layer.
2. The DNA hydrogel based on the signal amplification of the biomimetic enzymes
according to claim 1, characterized in that, the biomimetic enzymes are Au/Pt TNPs
formed by compounding Au and Pt, or the biomimetic enzymes are Cu/Au/Pt TNPs
formed by compounding Cu with Au and Pt.
3. The DNA hydrogel based on the signal amplification of the biomimetic enzymes
according to claim 1, characterized in that, the straight-chain polymer backbone is a
straight-chain polyacrylamide backbone, a straight-chain carboxymethyl cellulose
backbone, a straight-chain N-isopropylacrylamide backbone or a straight-chain polyvinyl
alcohol backbone.
4. The DNA hydrogel based on the signal amplification of the biomimetic enzymes
according to claim 1, characterized in that, a sequence of the MC-LR aptamer is: 5'-GGC
GCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC
CAT CTC CGC-3'; a high-affinity region of the sequence of the MC-LR aptamer is
selected to design the first short-chain DNA and the second short-chain DNA that are
partially complementary to the aptamer; and a sequence of the high-affinity region of the
MC-LR aptamer is: 5'-A TACCACCTCATTATGCCCCATCTCCGC-3'.
5. The DNA hydrogel based on the signal amplification of the biomimetic enzymes
according to claim 4, characterized in that, a sequence of the first short-chain DNA is:
'-Acrydite-TTT TTT GGG CAT AAT GAG-3'; and a sequence of the second short-chain
DNA is: 5'-Acrydite-TTT TTT GGG CAT AAT GAG-3'.
6. A reagent composition for detecting microcystin-LR, characterized by comprising:
the DNA hydrogel based on the signal amplification of the biomimetic enzymes according
to any one of claims 1-5, a peroxide oxidizer and a reductive chromogenic agent.
7. The reagent composition according to claim 6, characterized in that, the peroxide
oxidizer is H202and the reductive chromogenic agent is tetramethylbenzidine TMB.
8. A method for detecting microcystin-LR, characterized by comprising the
following steps:
Step 1: adding a certain amount of the DNA hydrogel based on the signal
amplification of the biomimetic enzymes according to any one of claims 1-5 to a sample
solution to be detected, and incubating the sample solution at 30-40°C for 0.5-2 h;
Step 2: adding hydrogen peroxide H202 and the chromogenic agent TMB to a
mixing system, adjusting pH with a buffer solution to weak acidity, reacting at a room
temperature, and then adding concentrated hydrochloric acid to terminate the reaction; and
Step 3: performing quantitative determination using a colorimetric detection
method.
9. The method for detecting the microcystin-LR according to claim 8, characterized
in that, in Step 3, an absorbance value at a specific wavelength is measured using an
ultraviolet-visible spectrophotometer; and a standard curve is drawn according to a series
of standard solutions with known concentrations for quantitative determination.
10. A preparation method of a DNA hydrogel based on signal amplification of
biomimetic enzymes, characterized by comprising the following steps:
Sl: preparing biomimetic enzymes Cu/Au/Pt TNPs having a peroxidase activity:
mixing a soluble copper salt and a complexing agent in water; then adding a
sufficient amount of a reducing agent; stirring for reaction, and then adding an appropriate
amount of an auric acid or aurate and an appropriate amount of a platinic acid or platinate;
stirring again for reaction to prepare the biomimetic enzymes Cu/Au/Pt TNPs having the
peroxidase activity;
S2: preparing an aptamer as well as a first short-chain DNA and a second
short-chain DNA, wherein a sequence of an MC-LR aptamer is: 5'-GGC GCC AAA CAG GAC
CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3'; a
sequence of a first short-chain DNA is: 5'-Acrydite-TTT TTT GGG CAT AAT GAG-3';
and a sequence of a second short-chain DNA is: 5'-Acrydite-TTT TTT GTA ATT GTC
ATG-3', and
S3: preparing the DNA hydrogel based on the signal amplification of the
biomimetic enzymes, comprising the following sub-steps:
S31: dissolving the first short-chain DNA and the second short-chain DNA in a
buffer solution respectively to obtain a solution of the first short-chain DNA and a solution
of the second short-chain DNA;
S32: preparing a solution of a polymer monomer which can be polymerized to
generate a straight-chain polymer for DNA grating;
blending the solution of the first short-chain DNA with the solution of the
polymer monomer, and adding a polymerization initiator and a catalyst in a vacuum
environment to obtain a straight-chain polymer grafted with the first short-chain DNA;
blending the solution of the second short-chain DNA with the solution of the
polymer monomer, and adding a polymerization initiator and a catalyst in a vacuum
environment to obtain a straight-chain polymer grafted with the second short-chain DNA;
and
S33: mixing the straight-chain polymer grafted with the first short-chain DNA
and the straight-chain polymer grafted with the second short-chain DNA, adding the
biomimetic enzymes Cu/Au/Pt TNPs prepared in Step Sl, then adding an aptamer solution
acting as a cross-linking bridge, fully mixing for reaction, and cooling to a room
temperature to obtain the DNA hydrogel based on the signal amplification of the
biomimetic enzyme.
11. The preparation method according to claim 10, characterized in that, in Step S1,
the auric acid is a chloroauric acid; the aurate is chloroaurate; the platinum acid is
tetrachloroplatinic acid or hexachloroplatinic acid; and the platinate is tetrachloroplatinate
or hexachloroplatinate.
12. The preparation method according to claim 10, characterized in that, in Sub-step
S32, the solution of the polymer monomer is an acrylamide solution, the polymerization
initiator is an ammonium persulfate solution, and the catalyst is an
N,N,N,N-tetramethylethylenediaminesolution.
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