CN114527098A

CN114527098A - Construction and application of dual-mode fluorescence-colorimetric biosensor based on FMs and PMA

Info

Publication number: CN114527098A
Application number: CN202111502569.4A
Authority: CN
Inventors: 孟红敏; 李倩楠; 赵迪; 李朝辉
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2021-12-10
Filing date: 2021-12-10
Publication date: 2022-05-24

Abstract

The invention relates to the technical field of materials, in particular to preparation and application of a fluorescence-colorimetric dual-mode biosensor based on FMs and PMA, and provides a paper-based-smart phone colorimetric biosensor system for realizing rapid quantitative detection of ALP. The invention utilizes a fluorescence-colorimetric dual-mode sensor to detect the ALP content in serum, which comprises PMA, FMs, AAP, portable paper base and sample diluent. The fluorescence-colorimetric dual-mode sensor has good selectivity on ALP and low false positive, and improves the detection accuracy. The colorimetric and fluorescent detection limits of ALP are respectively 0.068U/L and 0.11U/L, and the detection ranges are respectively 0.2-30U/L and 0.2-35U/L, so that the content of ALP in serum can be accurately determined. The paper-based smart phone-based sensing system has good accuracy, the detection limit of ALP is 0.4U/L, the detection range is 2-50U/L, and the real-time detection of ALP in daily life can be realized.

Description

Construction and application of dual-mode fluorescence-colorimetric biosensor based on FMs and PMA

Technical Field

The invention relates to the technical field of materials, in particular to a fluorescence-colorimetric dual-mode biosensor based on FMs and PMA (phosphomolybdic acid) and based on the optical performance of Fluorescent Microspheres (FMs) and the wide ultraviolet absorption capacity and fluorescence quenching capacity of phosphomolybdic blue generated by reduction of phosphomolybdic heteropoly acid (PMA).

Background

The Fluorescent coded microspheres (FMs) refer to solid phase spheres which are formed by adsorbing or embedding Fluorescent substances on the surface or inside carrier particles by a physical method (adsorption method, embedding method, self-assembly method) or a chemical method (grafting method, copolymerization method), have diameters ranging from nanometer to micrometer (0.01-10 μ M), and can emit fluorescence under the excitation of an excitation light source. The microsphere is a novel functional microsphere, has the characteristics of large specific surface area, strong adsorbability, large agglutination, strong surface reaction capability and the like, has a stable morphological structure and stable and efficient luminous efficiency, so that the application of the microsphere in biomedical detection is very wide, and the related fields comprise biological analysis, disease diagnosis, immunoassay and the like.

FMs-based biosensing method belongs to fluorescence sensing method, and is an analysis detection tool widely applied in the fields of biology, chemistry and medicine. The biosensing method can realize the rapid detection of specific target analytes, and the sensitivity can reach 10⁹-10¹²The method is of order of magnitude, and has wide application in the fields of trace, microanalysis and the like in recent years. Since FMs can interact with many reagents and biomolecules to undergo fluorescence quenching or fluorescence enhancement, fluorescence intensity is often used as a reporter parameter for the detection signal of fluorescence sensing methods. Since there are many factors that cause a change in fluorescence intensity, all substances that affect the fluorescence property of FMs can be used as targets, and thus FMs-based analysis methods have been established.

In recent years, many studies have been made by domestic and foreign scholars on the method for measuring ascorbic acid by using heteropolyacid as a developer, and many of the methods require long-time heating in a water bath, which increases the complexity of the operation. Importantly, many saccharides and impurities with poor reducibility may interfere with the assay results under heating, thereby reducing the selectivity of the method. Therefore, the phosphomolybdic heteropoly acid (PMA) which can be rapidly reduced by Ascorbic Acid (AA) at normal temperature is a very significant detection strategy. Meanwhile, phosphomolybdic blue generated after PMA is reduced by ascorbic acid has a wide light absorption range and can absorb FMs fluorescence emission at 620nm, namely, FMs fluorescence quenching is carried out through an inner filtering effect, so that FMs can be free from a complex surface modification process when serving as a report signal.

Alkaline phosphatase (ALP) is a catalytic protein that catalyzes dephosphorylation of proteins, nucleic acids, and small molecules, is widely present in human tissues such as liver, bone, kidney, and placenta, plays a crucial role in regulating various intracellular metabolism, and is an important biomarker for many diseases. Previous studies have confirmed that hepatic dysfunction, bone damage, and the like are closely associated with abnormal ALP activity. The detection of ALP is very important for the diagnosis of bone diseases and can be applied therapeutically to hypophosphatemia patients lacking this enzyme, and the role in vascular calcification and as a treatment for sepsis-associated acute kidney injury are also research hotspots in recent years. Therefore, the development of an effective method for detecting ALP activity is of great significance for the diagnosis and treatment of related diseases.

The detection methods of ALP reported so far are very extensive, including colorimetry, fluorescence, electrochemical method, etc., in which ALP catalyzes hydrolysis of colorless p-nitrophenyl phosphate (NPP) to produce yellow p-nitrophenol, which is one of the commonly used methods for monitoring the activity of ALP. The method is simple to operate, has high efficiency, is clinically applied as a standard method, but has the defects of poor selectivity, limited linear range and the like, which seriously influences the application of the method in practice. Worse still, the p-nitrophenyl phosphate substrate is sensitive to light and can also hydrolyze in the absence of ALP, which can lead to false positive results. Therefore, further efforts are needed to develop a new strategy for detecting ALP activity with high practicability and high reliability.

With the increasing popularization of smart phones, smart phones are often applied to rapid on-site detection and analysis due to the advantages of portability, low cost, easy operation and the like. It is reported in the literature that many smartphone sensing methods for medical diagnosis have been developed, such as a blood type sensing method of reading a paper-based signal using a smartphone as a sensing method and a colorimetric sensing method of detecting a pH change of sweat and saliva. Smartphone-based fluorescence sensing methods have also been successfully used to detect specific nucleic acid sequences in liquid samples. The color information captured by the smart phone can be read by RGB color recognition software. Some smartphone-based algorithm software has also been developed to directly quantify color information in smartphone interfaces. Research has proved that the accuracy of colorimetric detection based on a smart phone can be compared favorably with that of a desk-top spectrometer.

Therefore, the fluorescence-colorimetric dual-mode biosensor constructed based on FMs and PMA can realize quick and accurate detection of ALP. At room temperature, PMA can be reduced by AA generated by the reaction of ALP and AAP to generate phosphomolybdic blue, and the phosphomolybdic blue quenches FMs fluorescence through an inner filtering effect to construct a fluorescence-colorimetric dual-mode biosensor to realize the sensitive detection of ALP. In addition, the instant detection and real-time monitoring of the biomarker ALP are realized by using a convenient sensing system for reading the colorimetric signal by a smart phone.

Disclosure of Invention

The invention provides a construction method and application of a fluorescence-colorimetric dual-mode biosensor based on FMs and PMA to overcome the problems of complex operation, poor accuracy, low sensitivity, easy occurrence of false positive and the like of the existing ALP detection technology, and realizes the sensitive detection of ALP. In addition, the convenient sensing system for reading the colorimetric signals by using the smart phone reads the optical signals on the reacted paper sheets, performs data analysis through RGB software, realizes instant detection and real-time monitoring of ALP, has the advantages of portability, low cost, easy operation and the like, and can be applied to rapid field detection and analysis.

The preparation method of the technology of the invention comprises the following steps:

construction and application of a fluorescence-colorimetric dual-mode biosensor based on FMs and PMA. The fluorescence-colorimetric dual-mode biosensing method comprises FMs, PMA, AAP and the like, wherein FMs has good optical performance and provides a fluorescence signal, and the diameter of the fluorescence-colorimetric dual-mode biosensing method is about 200 nm; the phosphomolybdic blue generated after PMA reduction has a wider light absorption range, FMs is subjected to fluorescence quenching through the inner filtering effect, a fluorescence-colorimetric dual-mode signal is output, and high-sensitivity detection of ALP in a buffer solution system is realized. FMs and PMA fluorescence-colorimetric dual-mode biosensor have colorimetric and fluorescence detection limits of 0.068U/L and 0.11U/L respectively for ALP. In addition, the rapid and convenient sensing system for reading the colorimetric signals by the smart phone realizes the real-time detection of the ALP of the biomarker, has good accuracy, has the detection limit of 0.4U/L, and is significant for realizing the real-time detection and real-time monitoring of the ALP in daily life.

The preparation and application of the fluorescent-colorimetric dual-mode biosensor based on FMs and PMA are as follows:

(1) a solution containing AAP and ALP (0-150U/L) was prepared in 2mL of CutSmart buffer, mixed, and reacted at 25 ℃ for 15 min.

(2) Adding PMA and FMs to the solution after the reaction in the step (1) at 25 ℃.

(3) And (3) reacting the reaction solution in the step (2) for 5min, and detecting the ultraviolet absorption intensity of the sample within the ultraviolet absorption scanning range of 500-900 nm.

(4) And (3) after the solution in the step (2) is reacted for 180min, detecting the fluorescence intensity of the sample under the conditions that the fluorescence emission wavelength lambda max is 625nm (the excitation light wavelength lambda ex is 365nm) and the emission peak range is 600-.

The CutSmart buffer solution in the step (1) is 50mM CH₃COOK+30mM Tris-CH₃COOH，10mM CH₃COOMg +100 μ g/mL bovine serum albumin pH 7.9; the concentration of the AAP solution is 5 mM; the reaction concentration of ALP was 0-150U/L.

The concentration of PMA in the step (2) is 2mM, and the concentration of FMs is 5 mu g/mL.

The construction and application of the portable paper-based colorimetric sensing system are as follows:

(1) and (3) putting the round absorption pad into a PMA solution prepared by ultrapure water, soaking for 5s, taking out, standing at room temperature, and naturally drying to obtain the portable paper base.

(2) Preparing 2mL of solution containing AAP and ALP by using CutSmart buffer solution, uniformly mixing, reacting for 15min, and dropwise adding the reacted solution onto the paper base prepared in the step (1).

(3) And (3) after 20min, placing the paper base processed in the step (2) under an indoor white fluorescent lamp, placing the smart phone right above the paper base for photographing, and then reading the photographed photo by using the smart phone APP capable of reading RGB numerical values.

The concentration of the PMA solution in the step (1) is 2 mM; FMs was at a concentration of 5. mu.g/mL.

The concentration of the AAP solution in the step (2) is 5 mM; the reaction concentration solution of ALP is 0-80U/L; the diameter of the circular absorption pad is 2 cm; dripping the solution on the air-dried paper base to obtain 10 mu L of the solution.

The invention has the following beneficial effects:

1. FMs in the present invention has excellent optical properties and is capable of interacting with a variety of reagents and biomolecules to cause fluorescence quenching or fluorescence enhancement. The phosphor-molybdenum blue generated by rapidly reducing PMA by AA has a wide absorption range and can provide a colorimetric signal, and FMs can be subjected to fluorescence quenching through an internal filtration effect, so that FMs can be free of a complex surface modification process when used as a fluorescent signal. The method has the advantages of simple operation, rapid detection and the like. The biosensor based on FMs and PMA is constructed, so that the biosensor based on FMs and PMA can provide double signals of colorimetry and fluorescence, and the detection accuracy and sensitivity are improved.

2. The convenient sensing system for reading the colorimetric signal by the smart phone realizes the instant detection of the biomarker ALP, and has good accuracy. The sensing system can meet the clinical requirement on ALP activity detection, and has significance for realizing real-time detection and real-time monitoring of ALP in daily life.

3. Compared with the traditional construction using FMs biosensor, the biosensor designed by the method can resist the interference of substances such as amino acids (Leu, Phe, Tyr and Cys), other molecules (UA and NADH), enzymes (GOx and HAase) and the like which exist in plasma at the same time, solves the problem of false positive in the traditional method, and improves the detection accuracy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Fig. 1 is a schematic diagram of the dual-mode sensor prepared in example 1 and a paper-based-smart phone colorimetric biosensing system for detecting ALP.

Fig. 2 is a graph showing a response spectrum of the dual mode sensor prepared in example 1 to ALP.

FIG. 3 is the experimental condition optimization-optimizing AAP concentration of the dual-mode sensor prepared in example 1.

FIG. 4 is a graph of experimental optimization of dual mode sensor prepared in example 1-optimization of PMA concentration and color reaction time.

FIG. 5 is a response curve of the dual mode sensor prepared in example 1 to ALP-PMA color reaction to ALP.

FIG. 6 is a response curve-FMs fluorescence quenching response-of the dual mode sensor prepared in example 1 to ALP.

FIG. 7 is a selective examination of the dual mode biosensor prepared in example 1.

Fig. 8 is a response curve of the paper-based colorimetric sensing system prepared in example 1 to ALP.

FIG. 9 is the stability of PMA solution of example 1 to investigate the stability of aqueous PMA solution

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

A preparation method of a fluorescence-colorimetric dual-mode biosensor based on FMs and PMA, and a paper-based-smart phone colorimetric biosensor system for realizing rapid quantification of ALP, wherein the preparation principle is shown in figure 1: the prepared AAP and ALP solutions were added to the CutSmart buffer solution, respectively, at room temperature and incubated for 15 min. PMA and FMs are added into the solution after the reaction to obtain the fluorescence-colorimetric dual-mode biosensor. The experimental procedure was as follows:

the colorimetric-fluorescent dual-mode biosensor comprises PMA, FMs, AAP and the like.

In the present invention, materials such as a colorimetric reaction developer PMA, a fluorescent reaction substrate FMs, and AAP are commercially available.

FMs and PMA fluorescence-colorimetric dual-mode biosensor were prepared as follows:

the solution containing 5mMAAP and 10U/L ALP is prepared by CutSmart buffer solution, mixed evenly and reacted for 15min at 25 ℃, 2mM PMA and 5 mu g/mL FMs are added into the reacted solution, the reaction is carried out for 5min at 25 ℃, the ultraviolet absorption scanning range is 500-900nm, and the ultraviolet absorption intensity of the sample is detected. After 180min of reaction, the fluorescence intensity of the sample was measured under the conditions of a fluorescence emission wavelength λ max of 625nm (excitation wavelength λ ex of 365nm) and an emission peak range of 600-675nm as shown in FIG. 2.

The construction of the portable paper-based colorimetric sensing method is as follows:

preparing 2mM PMA solution by using ultrapure water, putting a circular absorption pad with the diameter of 2cm into the PMA solution for soaking for 5s, taking out the circular absorption pad, standing for 30min at room temperature for naturally drying, preparing 2mL of solution containing 5mM AAP and ALP with a certain concentration by using a CutSmart buffer solution, uniformly mixing, reacting for 15min, dropwise adding 10 mu L of reacted solution onto a dried paper base, placing the paper base under an indoor white fluorescent lamp after 20min, placing a smart phone right above the paper base for photographing at the height of 30cm, and then reading the value of a photographed photo by using a smart phone APP with a RGB readable value.

As shown in FIG. 2, FMs, the ALP and AAP alone in the solution did not make PMA blue, but only AA was formed in the presence of both ALP and AAP. Significant quenching of FMs fluorescence occurs only when ALP and AAP co-exist to form AA, reducing PMA to phosphomolybdenum blue. It is demonstrated that the sensor is feasible to detect the target ALP. As can be seen from FIGS. 3 and 4, the ultraviolet absorption signal has a slow rising trend after the AAP concentration is greater than 5mM, the fluorescence signal is substantially stable after the AAP concentration is greater than 5mM, the reaction equilibrium time of the PMA solution with 2mM is less than that of the PMA solution with 1mM, and the fluorescence of the microsphere tends to be stable after 3 h. As can be seen from FIGS. 5 and 6, when the concentration of applied ALP is from 0.2 to 35U/L, the colorimetric signal becomes stronger and the fluorescent signal becomes weaker, indicating that the sensor can be used for detecting ALP. As can be seen from fig. 7, when the target ALP was present, more significant colorimetric and fluorescent signals could be observed, and hardly responded to other substances. The results demonstrate that the dual-mode sensor has good selectivity for ALP and can be used for specific determination of ALP activity. As shown in FIG. 8, different concentrations of ALP (0-80U/L) are measured by using a paper-based colorimetric sensing system, the shade of the phosphor-molybdenum blue color on the paper base is related to the active concentration of the ALP, and the rapid quantification of the ALP can be realized by the paper-based smart phone colorimetric biosensing system. As shown in FIG. 9, the prepared PMA was allowed to stand for 1-120 days, and the colorimetric signal output from the paper substrate was substantially stable. The results indicate that the PMA solution is capable of maintaining stable properties over a long period of time.

Examples of the effects of the invention

In example 1 of the application, the application of the FMs and PMA-based fluorescence-colorimetric dual-mode biosensor in detecting ALP in human serum comprises the following steps:

a1% fetal bovine serum solution was prepared using CutSmart buffer. Preparing 2mL of a solution containing 5mM AAP and different concentrations of ALP (0, 5, 10 and 20U/L) by using a 1% fetal bovine serum solution, uniformly mixing, reacting for 15min, adding the reacted solution into 2mM PMA and 5 mu g/mL FMs, reacting for 5min, recording an ultraviolet absorption spectrum of a product by using an ultraviolet-visible spectrophotometer, and recording a fluorescence emission spectrum of the product by using a fluorescence spectrophotometer after reacting for 180 min.

Preparing a 2mM PMA solution by using ultrapure water, putting a circular absorption pad with the diameter of 2cm into the PMA solution for soaking for 5s, taking out the circular absorption pad, standing for 30min at room temperature for naturally drying, preparing 2mL of a solution containing 5mM AAP and ALP (0-80U/L) with different concentrations by using a CutSmart buffer solution, uniformly mixing, reacting for 15min, dropwise adding 10 mu L of the reacted solution onto the dried paper base, placing the paper base under a white fluorescent lamp in a room after 20min, placing a smart phone right above the paper base for photographing at the height of 30cm, and reading the photographed photo by using a smart phone APP capable of reading RGB values. Solutions containing 5mM AAP and different concentrations of ALP (0, 5, 10, 20U/L) were prepared with 1% fetal bovine serum solution (CutSmart buffer) and ALP detection in serum samples was performed on paper substrates according to the procedure described above.

As can be seen from Table 1, the activity of ALP in 5% fetal calf serum was detected by the spiking recovery method, and considering that the normal level of ALP activity in normal human serum was in the range of 46-190U/L, the sensitivity of this method was able to detect ALP activity in 20-fold diluted serum. With satisfactory recovery. Thus, these results demonstrate that this sensing strategy is feasible in a complex biological environment with potential for clinical ALP activity monitoring.

As can be seen from Table 2, the detection of ALP activity in 5% fetal calf serum by the labeling recovery method has a satisfactory recovery rate, and the paper-mobile phone-based colorimetric biosensing system is feasible in serum samples and has potential application in clinical ALP activity monitoring.

TABLE 1 serum sample analysis for dual mode ALP sensing

TABLE 2 detection of ALP in serum of portable paper-based fetal calf

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. Construction and application of a fluorescence-colorimetric dual-mode biosensor based on FMs and PMA. The fluorescence-colorimetric dual-mode biosensing method comprises FMs, PMA, AAP and the like, wherein FMs has good optical performance, provides a fluorescence signal and has the diameter of 200 nm; the phosphomolybdic blue generated after PMA reduction has a wider light absorption range, FMs is subjected to fluorescence quenching through the inner filtering effect, a fluorescence-colorimetric dual-mode signal is output, and high-sensitivity detection of ALP in a buffer solution system is realized. FMs and PMA fluorescence-colorimetric dual-mode biosensor have colorimetric and fluorescence detection limits of 0.068U/L and 0.11U/L respectively for ALP. In addition, the rapid and convenient sensing system for reading the colorimetric signals by the smart phone realizes the real-time detection of the ALP of the biomarker, has good accuracy, has the detection limit of 0.4U/L, and is very significant for realizing the real-time detection and real-time monitoring of the ALP in daily life.

2. A colorimetric-fluorometric dual-mode biosensor according to claim 1, wherein the biosensor is selected from the group consisting of FMs and PMA: the FMs and PMA colorimetric-fluorescent dual-mode biosensor is prepared by the following steps:

(1) 2mL of AAP-and ALP-containing solution was prepared using CutSmart buffer, mixed and reacted at 25 ℃ for 15 min.

3. A colorimetric-fluorometric dual-mode biosensor according to claim 2, wherein said biosensor is selected from the group consisting of FMs and PMA: the CutSmart buffer solution in the step (1) is 50mM CH₃COOK+30mM Tris-CH₃COOH+10mM CH₃COOMg +100 μ g/mL bovine serum albumin (pH 7.9); the concentration of the AAP solution is 5 mM; the reaction concentration of ALP was 0-150U/L.

4. The method for colorimetric-fluorometric bimodal biosensing of FMs and PMA as defined in claim 2, wherein: the concentration of PMA in the step (2) is 2mM, and the concentration of FMs is 5 mu g/mL.

5. The paper-based-smart phone colorimetric biosensing method according to claim 1, characterized in that: the paper-based-smart phone colorimetric biosensing system is prepared as follows:

(3) And (3) after 20min, placing the paper base processed in the step (2) under an indoor white fluorescent lamp, placing the smart phone right above the paper base for photographing, and reading the value of the photo by using the smart phone APP capable of reading the RGB value.

6. The paper-based-smart phone colorimetric biosensing method according to claim 2, characterized in that: the concentration of the PMA solution in the step (1) is 2 mM; FMs was at a concentration of 5. mu.g/mL.

7. The paper-based-smart phone colorimetric biosensing method according to claim 2, characterized in that: the concentration of the AAP solution in the step (2) is 5 mM; the reaction concentration solution of ALP is 0-80U/L; the diameter of the circular absorption pad is 2 cm; 10 μ L of the solution was added dropwise to the air-dried paper base.

8. The colorimetric-fluorescent dual-mode biosensor method for determining alkaline phosphatase (ALP) based on Fluorescent Microspheres (FMs) and phosphomolybdic heteropoly acid (PMA) as claimed in any one of claims 1 to 7, and a paper-based-smartphone colorimetric biosensing method is proposed to achieve rapid quantitative detection of ALP, characterized in that: PMA can be rapidly reduced by AA generated by the reaction of ALP and AAP at room temperature to generate phosphomolybdic blue, the phosphomolybdic blue has ultraviolet absorption within 900nm of lambda 500-620 nm and can absorb FMs fluorescence emission at the lambda 620nm, so that FMs fluorescence is quenched by an inner filter effect, and the ALP is sensitively detected.