Label-free optical nano sensor for detecting activity of beta-glucosidase and application thereof
阅读说明：本技术 β-葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用 (Label-free optical nano sensor for detecting activity of beta-glucosidase and application thereof ) 是由 刘自平 刘莎莎 田野 周帅 于 2021-09-03 设计创作，主要内容包括：本发明公开了一种β-葡萄糖苷酶活性检测用免标记型光学纳米传感器及应用,该传感器中蛋白-无机杂交纳米花可通过电子转移将荧光红染料催化转化为深紫红色,具有强荧光的试卤灵。当向体系中加入β-Glu时,由于生氰苷的存在,β-Glu能够特异性水解生氰苷,并生成CN～(-)。CN～(-)能够有效地抑制蛋白-无机杂交纳米花的催化活性,使得体系的荧光强度减弱,同时伴随着溶液颜色变浅。通过监测体系的颜色变化以及荧光的“Turn on-off”,可以实现β-Glu的光学双信号检测。所构建荧光纳米传感器还可用于土壤中β-Glu的定量检测。此外,该传感器在筛选β-Glu抑制剂方面具有一定的应用潜力。(The invention discloses a label-free optical nano-sensor for detecting beta-glucosidase activity and application thereof, wherein a protein-inorganic hybrid nano-flower in the sensor can catalyze and convert a fluorescent red dye into deep purple red through electron transfer, and has strong fluorescent resorufin. When beta-Glu is added to the system, beta-Glu is capable of specifically hydrolyzing cyanogenic glycoside due to the presence of cyanogenic glycoside and generating CN ‑ 。CN ‑ Can effectively inhibit the catalytic activity of the protein-inorganic hybrid nano flower, so that the fluorescence intensity of the system is weakened, and the color of the solution is lightened. By monitoring the color change of the system and the fluorescent 'Turn on-off', the optical double-signal detection of the beta-Glu can be realized. The constructed fluorescent nano sensor can also be used for quantitative detection of beta-Glu in soil. In addition, the sensor has certain application potential in the aspect of screening the beta-Glu inhibitor.)
1. A label-free optical nano sensor for detecting the activity of beta-glucosidase is characterized by comprising the following components: protein-inorganic hybrid nano flower with enzyme-imitating activity, fluorescent red dye, cyanogenic glycoside and hydrogen peroxide solution.
2. The label-free optical nanosensor for detecting β -glucosidase activity as claimed in claim 1, wherein the protein-inorganic hybrid nanoflower with mimic enzyme activity is BSA-Cu3(PO4)2·3H2O NFs。
3. The label-free optical nanosensor for detecting β -glucosidase activity as claimed in claim 1 or 2, wherein the concentration of the fluorescent red dye is 20 μmol-L-1。
4. The label-free optical nanosensor for detecting β -glucosidase activity as claimed in claim 1 or 2, wherein the cyanogenic glycoside is amygdalin solution.
5. The label-free optical nanosensor for detecting β -glucosidase activity as claimed in claim 4, wherein the concentration of the amygdalin solution is 20 mmol-L-1。
6. The label-free optical nanosensor for detecting β -glucosidase activity as claimed in claim 1 or 2, wherein the concentration of hydrogen peroxide solution is 200 mmol-L-1。
7. The application of the label-free optical nano sensor for detecting the activity of the beta-glucosidase is characterized by being used for detecting the beta-Glu in soil and screening a beta-Glu inhibitor.
Technical Field
The invention relates to the technical field of beta-glucosidase activity detection, in particular to a label-free optical nano sensor for beta-glucosidase activity detection and application thereof.
Background
Beta-glucosidase (beta-Glu), a glycosyl hydrolase capable of specifically catalyzing hydrolysis of beta-glycosidic bonds, has important significance for plants, animals and microorganisms to generate functional secondary metabolite glucoside. In the last two decades, beta-Glu has been widely used in the fields of medicine, bioenergy, environment, food, etc. For example, levels of β -Glu activity are associated with metabolic diseases such as diabetes, bacterial or viral infections, and cancer [ lillulund, v.h.; jensen, h.h.; liang, x.; bols, M.Recent definitions of transition-state analogue glycosylation inhibitors of non-natural product orientation chem Rev,2002,102,515-553 ]. In biotechnology, β -Glu activity plays an extremely important role in the production of second and third generation environmentally friendly biofuels and chemicals from renewable lignocelluloses [ Huang, d.l.; zeng, g.m.; feng, c.l.; hu, s.; jiang, x.y.; tang, l.; su, f.f.; zhang, y.; zeng, w.; liu, H.L.Degradation of lead-associated lipolytic waste by chromatography of lysine and the reduction of lead susceptibility. environ Sci Technol,2008,42,4946 and 4951. In the aspect of environment, research shows that the activity of beta-Glu in eutrophic lakes is closely related to the mass propagation of phytoplankton bloom in spring [ Chr Louist, R.J.; arch Hydrobiol Beih Ergebn Limnol,1990,34,93-98 ]. In agriculture, there has been an exciting finding in recent years that β -Glu activity can be used as a biological indicator of soil quality [ Sazawa, k.; kuramitz, H.Hydrodynamic volumetric as a rapid and simple method for evaluating soil enzyme activities, Sensors,2015,15, 5331-. Therefore, the detection of the beta-Glu and the monitoring of the activity of the beta-Glu have important meanings.
At present, the conventional method for measuring the activity of beta-Glu is based on the conversion of a substrate (p-nitrophenyl-beta-D-glucopyranoside) into p-nitrophenol followed by spectrophotometric quantification. However, an important drawback of this method is that the substrate used for detecting β -Glu activity is very unstable; in addition, the method involves a large number of very environmentally harmful agents [ step, p.w.; messina, g.a.; bianchi, g.; olsina, r.a.; raba, J.determination of beta-glucosidase activity in fluids with a biological sensor modified with multi walled carbon n nanoparticles, anal biological Chem,2010,397, 1347-. Recently, some of the reported novel methods for fluorescence detection of β -Glu activity are mostly based on β -glycosides of phenol derivatives, such as nitrophenol [ Yan, s.; wu, G.prediction of microfastener-resistant of beta-glucosides using nitrophenyl-beta-D-glucopyranoside as substrate. protein peptide Lett,2011,18, 1053-; messina, g.a.; bianchi, g.; (iii) Olsina, R.A. determination of the β -glucosidase activity in differential enzymes by pre-mammalian enzyme assay using mammalian electrophoresis with laser-induced fluorescence detection. J.Fluoresc, 2010,20,517-523] and 7-hydroxycoumarin [ Watanabe, A.; suzuki, m.; ujiie, s.; gomi, K.Purification and enzymic chromatography of alpha novel beta-1, 6-glucosidases from Aspergillus oryzae.J. Biosci Bioeng,2016,121, 259-264. However, the blue fluorescence of the fluorophore used in such methods overlaps with the background of the biological sample, and it is necessary to isolate and purify it before measuring the enzyme activity, resulting in complicated analysis of the β -Glu activity. In addition, the time-consuming or poor reproducibility of detection also limits the application of these detection methods.
Therefore, the design of a beta-Glu activity detection reagent which is simple to operate, high in sensitivity, effective, low in cost and environment-friendly is urgently needed.
Disclosure of Invention
In view of the above, the invention provides a label-free optical nanosensor for detecting beta-glucosidase activity and an application thereof, so as to solve the problems of complex operation, poor effectiveness and the like in the conventional beta-glucosidase activity detection.
In one aspect, the invention provides a label-free optical nano-sensor for detecting beta-glucosidase activity, which comprises the following components: protein-inorganic hybrid nano flower with enzyme-imitating activity, fluorescent red dye, cyanogenic glycoside and hydrogen peroxide solution.
Preferably, the protein-inorganic hybrid nano flower with the enzyme imitating activity is BSA-Cu3(PO4)2·3H2O NFs。
Further preferably, theThe concentration of the fluorescent red dye is 20 mu mol.L-1。
More preferably, the cyanogenic glycoside is amygdalin solution.
More preferably, the concentration of the amygdalin solution is 20 mmol.L-1。
More preferably, the concentration of the hydrogen peroxide solution is 200 mmol.L-1。
On the other hand, the invention also provides application of the label-free optical nano sensor for detecting the activity of the beta-glucosidase, and particularly relates to application of the label-free optical nano sensor to detection of beta-Glu in soil and screening of a beta-Glu inhibitor.
The label-free optical nano sensor for detecting the activity of the beta-glucosidase, provided by the invention, can be used for catalytically converting a fluorescent red dye into deep purple red Resorufin (Resorufin) with strong fluorescence by virtue of protein-inorganic hybrid nano flowers in the sensor through electron transfer. When beta-Glu is added to the system, the beta-Glu can specifically hydrolyze cyanogenic glycoside due to the presence of cyanogenic glycoside and generate cyanide ion (CN)-)。CN-Can effectively inhibit the catalytic activity of the protein-inorganic hybrid nano flower, so that the fluorescence intensity of the system is weakened, and the color of the solution is lightened. CN-The concentration of the beta-Glu is controlled by the enzymolysis reaction triggered by the beta-Glu with different concentrations, so that the optical double-signal detection of the beta-Glu can be realized by monitoring the color change of a system and the fluorescent 'Turn on-off'. Under the optimal condition, the constructed optical nano-sensor is 0.5-1500 U.L-1The detection of beta-Glu shows good linear relation; the detection limit is 0.33 U.L-1. The constructed fluorescent nano sensor can also be used for quantitative detection of beta-Glu in soil. In addition, the sensor has certain application potential in the aspect of screening the beta-Glu inhibitor.
Compared with the prior art, the optical nano-sensor established by the invention is based on the protein-inorganic hybrid nano-flower with the enzyme-like activity, so that the constructed sensor has the advantages of simple preparation, low cost, strong catalytic activity, difficult influence by environmental conditions and the like. In addition, the optical nano-sensor does not need to be marked, and only utilizes electron transfer induced fluorescence quenching and special enzymolysis induced fluorescence recovery to trigger optical response, so that the multi-step functionalization or time-consuming and labor-intensive complex marking process is avoided. Moreover, the optical sensor realizes dual-signal detection, so that the optical sensor has a more potential application prospect. The fluorescent nano sensor has a wider linear response range on the activity of beta-Glu, and the detection limit is lower.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 shows the self-assembly synthesis of BSA-Cu according to the present invention3(PO4)2·3H2O NFs，(B)H2O2BSA-Cu in the Presence of3(PO4)2·3H2O NFs catalyze and oxidize Amplex Red into resorufin, (C) a schematic diagram of a label-free dual-signal optical nanosensor for detecting the activity of beta-Glu;
FIG. 2 shows the blank AR (a), BSA-Cu3(PO4)2·3H2O NFs + AR mixed solution (b), H2O2+ AR mixed solution (c), AR + BSA-Cu3(PO4)2·3H2O NFs+H2O2Mixed solution (d), and AR + BSA-Cu3(PO4)2·3H2O NFs+H2O2(iii) fluorescence emission spectra of each of the + β -Glu + Amy mixed solutions (e). The color of the solution (a), (b), (c), (d) and (e) is shown in the insetAnd (4) color. FL is an abbreviation for fluorescence, AR is an abbreviation for Amplex Red;
FIG. 3 shows BSA-Cu of the present invention3(PO4)2·3H2O NFs and Amplex Red mixed solution at different concentrations of H2O2A fluorescence emission spectrogram (A) in the presence of (0-200.0 mmol. L-1); different concentrations of H2O2The dotted line relationship between the fluorescence intensity of the mixed solution is shown in the figure, and the inset is H in the range of 5-60 mmol.L-12O2A linear relationship (B) between the concentration and the fluorescence intensity of the system;
FIG. 4 is a graph of incubation time versus BSA-Cu prepared3(PO4)2·3H2Influence of the catalytic performance of O NFs;
FIG. 5 is pH vs. BSA-Cu3(PO4)2·3H2Effects of O NFs and HRP catalytic activity;
FIG. 6 shows a BSA-Cu based assay according to the present invention3(PO4)2·3H2O NFs-H2O2-Amplex Red-Amy nanosensor responding to different concentration beta-Glu (0-1500.0 U.L)-1) Fluorescence emission spectrum of (a); a linear relation graph between the concentration of beta-Glu and the fluorescence intensity ratio (I/I0) of the system, wherein I and I0 represent BSA-Cu in the presence or absence of beta-Glu3(PO4)2·3H2O NFs-H2O2-fluorescence emission spectrum (b) of Amplex Red-Amy mixed solution; corresponding color change graphs (c) of different beta-Glu activity catalytic sensing systems under sunlight;
FIG. 7 shows a BSA-Cu based assay according to the present invention3(PO4)2·3H2O NFs-H2O2And (4) researching the anti-interference capability and selectivity of the Amplex Red-Amy optical nano sensor on beta-Glu detection.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, but the present invention is not limited thereto.
All chemicals and reagents in the following examples were at least of analytical grade, all ultrapure water (18.2 M.OMEGA.. multidot.cm) used in the experiments-1) All from Milli-Q Integral 10 water purification unit.
Example 1: BSA-Cu3(PO4)2·3H2Preparation of O NFs
BSA-Cu3(PO4)2·3H2The specific synthetic route of O NFs is as follows [ see FIG. 1(A)]: first, 200 mmol. L-1CuSO4Solution infusion 1690. mu.L solution containing 0.1 mg/mL-150 mmol. L of BSA-1In Phosphate Buffered Saline (PBS) at pH 6.8, and then left to stand at room temperature for 12 hours. BSA-Cu to be prepared3(PO4)2·3H2The O NFs were centrifuged at 10000rpm for 10min and then washed 3 times with ultrapure water for purification. Finally, the collected blue-green precipitate was redispersed in 200. mu.L PBS and stored at 4 ℃ for use in subsequent experiments.
Example 2: BSA-Cu3(PO4)2·3H2Research on catalytic performance of O NFs
By means of H2O2And Amplex Red solution (fluorescent and chromogenic substrates) for BSA-Cu3(PO4)2·3H2The peroxidase-mimetic properties of O NFs. As shown in FIG. 2, in the control experiment, Amplex Red solution (curve a), Amplex Red and NFs mixed solution (curve b) and Amplex Red and H2O2The mixed solution (curve c) has no obvious fluorescence emission peak between 565 nm and 800 nm. When BSA-Cu is added3(PO4)2·3H2O NFs, Amplex Red and H2O2When the three were mixed, the mixed solution showed a significant increase in fluorescence at 584nm (curve d), which was derived from the oxidation product resolufin of Amplex Red, and the background fluorescence was very low and negligible. The above results show that BSA-Cu3(PO4)2·3H2O NFs have excellent activity in mimicking peroxidase. When the excitation wavelength is 550nm, the maximum fluorescence emission wavelength of the reaction system is about 584 nm. At the same time, as shown in the inset of fig. 2, the solution was observed to rapidly change from colorless to a typical deep purple color, which was easily discernible to the naked eye.
To further study BSA-Cu3(PO4)2·3H2Catalytic performance of O NFs, we tested a certain amount of BSA-Cu3(PO4)2·3H2O NFs, Amplex Red with varying concentrations of H2O2Fluorescence emission spectrum of the system when the solution coexists. As shown in FIG. 3, in the range of 0.05-200 mmol. multidot.L-1In the concentration range, with H2O2The fluorescence emission intensity of the system is gradually enhanced when the concentration is increased; as can be seen from the inset in FIG. 3, the fluorescence emission intensity of the system is related to H2O2The concentrations exhibited a good linear relationship. The above results further confirm that BSA-Cu3(PO4)2·3H2O NFs have good peroxidase-like catalytic properties.
It is well known that the catalytic properties of the enzyme are closely related to the reaction conditions, in particular pH and incubation time, and therefore this example examines the pH and incubation time of the solution versus the BSA-Cu prepared3(PO4)2·3H2Influence of the enzymatic properties of O NFs. As can be seen from FIG. 4, with Amplex Red as the substrate, BSA-Cu in acidic medium was observed at pH range 4.0-9.03(PO4)2·3H2The catalytic activity of O NFs and the catalytic activity of horseradish peroxidase (HRP) are higher than that of an alkaline medium, but BSA-Cu3(PO4)2·3H2O NFs have generally better catalytic performance than HRP. As can be seen from FIG. 5, in a certain range, the fluorescence emission intensity of the system is enhanced along with the increase of the incubation time, and reaches a platform in about 90min, which indicates that the catalytic reaction is completed in 90 min.
Example 3: construction of optical nano-sensor and response thereof to beta-Glu
First, 20000 U.L was prepared-1The stock solution of beta-Glu of (1). Separately, 150. mu.L of NFs solution and 50. mu.L of amygdalin solution (20 mmol. L)-1) A series of 50. mu.L of β -Glu solutions of different concentrations were added and mixed well and incubated at room temperature for 1h for enzymatic reaction. Then, 20. mu. mol. L of each of the above-mentioned reaction solutions was added-1Amplex Red solution and 200 mmol. L-1H of (A) to (B)2O2The solution is prepared by mixing a solvent and a solvent,using phosphate buffer solution (10 mmol. L)-1pH 6.8) to 1.5 mL. After mixing well, incubation was carried out at room temperature for 90 minutes. The fluorescence emission spectrum of the mixed solution in the range of 565-800nm under the excitation light condition of 550nm was measured, and the result is shown in FIG. 6. The excitation and emission slits are both 5 nm. It can be observed from FIG. 6 that the concentration is 0 to 1500.0 U.L-1In the range, BSA-Cu increases with the amount of beta-Glu3(PO4)2·3H2O NFs-H2O2The fluorescence intensity of the Amplex Red-Amy system at 584nm decreases gradually, and the color of the mixed solution gradually changes from dark purple to light magenta [ FIG. 6(c) ]]. In addition, it can be estimated from FIG. 2 that BSA-Cu3(PO4)2·3H2O NFs-Amplex Red-H2O2The fluorescence emission intensity at 584nm of the system dropped to 33.8% of the original intensity. Meanwhile, FIG. 6(b) shows that the concentration of. beta. -Glu is in the range of 0.5-1500 U.L-1Ratio of in-range to fluorescence intensity (I/I0, I and I0 represent BSA-Cu in the absence or presence of β -Glu3(PO4)2·3H2O NFs-H2O2Fluorescence emission spectrum of Amplex Red-Amy mixed solution) has good linear relation, and the linear regression equation is that I/I0 is 0.992-0.0002[ beta-Glu [ ]](U·L-1)(R20.997). The detection Limit (LOD) of beta-Glu is 0.33 U.L-1. Compared with the existing beta-Glu detection method, the method has lower LOD and wider quantitative range.
Example 4: selective investigation of optical nanosensors
Selectivity is an important parameter in evaluating the performance of a new sensor, and particularly for sensors with potential application value in complex environmental samples, highly selective response to a target is necessary. Therefore, adding Na+、K+、Ca2 +、Mg2+、NO3-And Cl-1Under the condition of a plurality of common coexisting substances such as glucose oxidase (Glu), Glucose Oxidase (GOX), Tyrosinase (TYR), acid phosphatase (ACP), L-cysteine, glucose, protamine and the like, the optical nano-sensor is subjected toThe results of the selectivity experiments are shown in FIG. 7. The concentration of ACP was 1.0. mu.U.mL-1. GOX concentration of 1.0. mu.g/mL-1. The concentration of L-cysteine, glucose and protamine is 1.0. mu. mol. L-1。Na+、K+、Ca2+、Mg2+、NO3-And Cl-1Has a concentration of 1.0. mu. mol. L-1. As can be seen from fig. 7, the inventive optical nanosensor is not only non-responsive to other possible coexisting substances, but also selective to β -Glu in the presence of other possible coexisting substances, indicating that the optical nanosensor has strong anti-interference capability.
Example 5: detection of beta-Glu activity in soil solution
The soil used in the present embodiment is provided by the local biogeochemical laboratory, and all soils are topsoil (0-20 cm). After removing roots and other impurities from the plants in the soil sample, the soil sample was sieved through a 2mm sieve. Dissolving 1.0g of a soil sample in 15mL of ultrapure water, and shaking for 1 hour at room temperature; then, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was filtered. Various concentrations of β -Glu were added to soil samples to prepare additive samples, and then β -Glu activity in the soil samples was measured according to the procedure described in example 3, with the results shown in table 1. And (3) carrying out a recovery rate test by adopting a standard sample adding method, deducing the activity of the beta-Glu in the sample according to a standard curve and a regression equation, and calculating the RSD from 3 parallel samples generally. As can be seen from Table 1, the average recovery of β -Glu from spiking was 96.2-104% with RSD less than 5.0%. Thus, the accuracy and precision of the described sensing system is satisfactory.
Table 1: detection result of optical nano sensor in actual soil sample
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
It is to be understood that the present invention is not limited to what has been described above, and that various modifications and changes may be made without departing from the scope thereof. The scope of the invention is limited only by the appended claims.