CN111635532B - Aggregation-induced fluorescence guest functionalized composite infinite coordination polymer nanoparticle and preparation and application thereof - Google Patents
Aggregation-induced fluorescence guest functionalized composite infinite coordination polymer nanoparticle and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses composite infinite coordination polymer nanoparticles (composite ICPs) for aggregation-induced fluorescence guest functionalization and a preparation method thereof. The composite ICPs comprise a host and a guest, and can simultaneously present Monomer Emission (ME) and aggregate emission (AIE) effects under the regulation and control of the host and the guest. The invention also discloses a double-proportion type fluorescence visualization analysis method for the anthrax biomarker 2, 6-dipicolinic acid (DPA). In addition, by utilizing the morphological change of the compound ICPs during the stimulus response period, the coffee ring deposition morphology is regulated and controlled through the stimulus response of the compound ICPs, and the coffee ring deposition morphology is expanded into a new signal reading channel.
Description
Technical Field
The invention belongs to the field of nano material preparation and detection, and relates to a double-proportion type fluorescence visualization analysis method based on a gathering induction light-emitting object functionalized infinite coordination polymer, namely a multichannel coffee ring test paper and application thereof in field analysis of 2, 6-dipicolinic acid.
Background
From 20Bacillus anthracis (b.anthracosis) spores have received widespread attention worldwide as a potential terrorist attack agent since 01. The bacillus anthracis has strong vitality and wide infection route, can survive in soil for decades, has nondecreasing pathogenicity, has high lethality rate almost all over the world, and particularly has great harm to social public health and economic development due to the anthrax caused by the bacillus anthracis. Therefore, the development of a reliable method for on-site detection of the anthrax contamination biomarker dipicolinic acid (DPA) is of great significance for controlling the outbreak and prevention of anthrax. Currently, detection of DPA is mostly based on lanthanide metal ions (Ln)3+) The developed analysis method, but most of the analysis methods are single-channel fluorescence response mode or internal reference fluorescence response mode, on one hand, the method is easily influenced by environmental fluctuation to generate false positive results; on the other hand, under the excitation of an ultraviolet lamp, the resolution of the change of the fluorescence color is low, the quantitative analysis of the DPA depends on a large-scale laboratory optical instrument, and the practical application of the field analysis is limited to a certain extent. Due to the increasing need for rapid response to control anthrax outbreaks and to prevent bioterrorism attacks, significant improvements are needed not only in the research of the sensing mechanisms of the methods, but also to ensure the reliability of the methods in complex environmental systems.
In recent years, Infinite Coordination Polymers (ICPs) have attracted increasing attention as a new class of metal organic coordination polymer nanomaterials. The space network structure of the ICPs has high flexibility, and various objects with different optical properties can be wrapped in the host ICPs network structure through in-situ self-assembly wrapping performance, so that the composite ICPs nano particles with the host-object dual optical advantages are obtained. In addition, what is more important is that the flexibility of the structure enables the composite ICPs to respond to external stimuli quickly, and a foundation is provided for designing a quick double response based on the stimuli-responsive composite ICPs nano particles and further designing a new visual sensing mechanism. At present, most of composite ICPs nano particles have lanthanide series metal ions (Ln-ICPs) as central metal ions of host ICPs, have large Stokes shift and wide emission waveband, have high requirements on spectral conditions of object fluorescent molecules in order to realize dual response, and most of objects meeting the spectral conditions are fluorescent molecules or fluorescent nano materials with aggregation-induced fluorescence quenching effect (ACQ), so that on one hand, the selection of host and object materials of the composite ICPs nano particles and the development of a new sensing mechanism are limited depending on an object ACQ effect sensing mode; on the other hand, in the process of test paper formation or chip formation depending on the guest ACQ effect, the concentration of the sample is susceptible, which limits practical application. Therefore, the development of a brand new visual sensing mechanism and a visual analysis method by using a novel object with an anti-ACQ effect is of great significance.
The aggregation-induced emission (AIE) phenomenon, which was proposed from 2001 to now, brings new opportunities to the field of optical analysis as it greatly solves the problems faced by the conventional ACQ fluorescent probe molecules. In contrast to the optical response mode of ACQ fluorescent molecules, the AIE fluorescent molecules emit weak light even in a dilute solution state, but emit bright fluorescence when they are aggregated in a solution or in a solid state. Therefore, fluorescent molecules with AIE phenomenon are introduced into an ICPs host network structure as objects, the AIE effect of the objects is regulated and controlled through the stimulus response of the composite ICPs nanoparticles, and a brand new thought is provided for establishing a novel visual sensing mechanism and a novel method based on the stimulus response type composite infinite coordination polymer nanoparticles.
Coffee Ring Effect (CRE), a phenomenon frequently observed in everyday life, is a phenomenon in which the evaporation rate at the edge of a droplet is greater than that at the center, so that an outward capillary flow is generated inside the droplet and suspended particles are carried to the edge of the droplet and deposited in a ring shape at the edge. The system is simple and convenient, the required sample amount is small, the whole detection process only depends on the normal evaporation process, and great advantages are shown in portable and high-flux field detection. In 2005, a research group led by professor of the cell control institute of los angeles, university of california, academy of the american academy, used the coffee ring effect for chromatography at the nanoscale to achieve separation of nanoparticles of different sizes. For the composite ICPs nano-particles, after the stimulation response occurs, the change of the spatial structure of the composite ICPs nano-particles causes the change of the size of the nano-particles, and simultaneously, the number of the composite ICPs nano-particles with large particle size is reduced, and the number of the guest nano-particles or small molecules with small particle size is increased. Because the coffee ring effect generally exists in systems such as small molecules, small-particle-size noble metal particles, polymer nanoparticles and the like, the size-dependent nano chromatographic separation phenomenon of the coffee ring effect is combined with the size change of the composite ICPs nanoparticles before and after the stimulation response, the signal reading mode is expected to be further expanded through the changes of the size of the coffee ring spot ring, the ring diameter and the like, and a novel multichannel sensing mechanism and a novel strategy based on coffee ring effect regulation are developed.
So far, a material with aggregation-induced emission performance is selected as an object, the host-object effect is reasonably designed, a composite ICPs nano material which can simultaneously show ME and AIE effects is prepared, the metal competitive coordination effect is further regulated and controlled, the Monomer Emission (ME) and the aggregate fluorescence emission (AIE) of the object are further changed, a new multi-response visual sensing mechanism is constructed, and a more reliable new proportional fluorescence visual analysis method is developed; the stimulation response of the composite ICPs is combined with the coffee ring effect, the morphology of the coffee ring deposit is regulated and controlled through the changes of the size and the number before and after the stimulation response of the composite ICPs nano particles, the coffee ring deposit is converted into a new signal reading mode, a multi-channel sensing mode is developed by combining the change of fluorescence color, low-cost test paper is developed, and the field analysis aiming at the 2, 6-dipicolinic acid is really realized by combining an intelligent mobile phone provided with image recognition and processing software, which is not reported yet.
Disclosure of Invention
To overcome the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide aggregation-induced fluorescent guest-functionalized composite infinite coordination polymer nanoparticles (composite ICPs) that exhibit both ME and AIE effects. Further, by regulating and controlling the metal competitive coordination effect, the Monomer Emission (ME) and the aggregate fluorescence emission (AIE) of the object are changed, a new multi-response visual sensing mechanism is constructed, and a more reliable new double-proportion type fluorescence visual analysis method is developed; the stimulation response of the composite ICPs is combined with the coffee ring effect, the morphology of the coffee ring deposit is regulated and controlled through the changes of the size and the number before and after the stimulation response of the composite ICPs nano particles, the coffee ring deposit is converted into a new signal reading mode, a multi-channel sensing mode (method) is developed by combining the change of fluorescence color, low-cost test paper is developed, and the field analysis of the 2, 6-dipicolinic acid is really realized by combining a smart phone with image recognition and processing software.
The invention provides composite infinite coordination polymer nanoparticles (composite ICPs) for aggregation-induced fluorescence guest functionalization, which specifically comprise a host and a guest.
Wherein, the main ICPs are selected from ICPs formed by freely coordinating and combining any metal ions and corresponding ligands.
The metal ions are lanthanide metal ions (Ln-ICP) as central ions, including europium ions (Eu)3+) Terbium ion (Tb)3+) Cerium ion (Ce)3+) Lanthanum ion (La)3+) Neodymium ion (Nd)3+) Gadolinium (Gd)3+) Erbium ion (Er)3+) Ytterbium ion (Yb)3+) One or more of the following; preferably, it is europium ion (Eu)3+)。
The ligand is an alkaloid ligand, and comprises one or more of guanosine-5 '-monophosphate (GMP), adenosine-5' -monophosphate (AMP), uridine-5 '-monophosphate (UMP), cytidine-5' -monophosphate (CMP), guanosine-5 '-diphosphate (GDP), adenosine-5' -diphosphate (ADP), uridine-5 '-diphosphate (UDP), cytidine-5' -diphosphate (CDP), guanosine-5 '-triphosphate (GTP), adenosine-5' -triphosphate (ATP), uridine-5 '-triphosphate (UTP), cytidine-5' -triphosphate (CTP), and the like; preferably, guanosine-5' -monophosphate (GMP).
Further, the main body Ln-ICP of the composite infinite coordination polymer nano particle is Eu/GMP ICP, wherein GMP is a network structure formed by coordination of N7-C8 on a ligand and O on a phosphate group and europium ions.
The guest is a guest with aggregation-induced emission effect, and is one or more of water-soluble hydrophilic group functionalized tetraphenylethylene and derivatives thereof, including hydrophilic groups such as various sulfonates, carboxylates and quaternary ammonium salts, and the like, which show AIE in a host Ln-ICP (Ln-ICP); preferably, tetrakis (4-sulfophenyl) ethylene sodium salt (WSSu-TPE).
Wherein the molar ratio of the metal ions, the ligand and the guest is 37:37: (1-10); preferably, 37:37: 1.
The guest molecules are wrapped in the host through the interaction of the host and the guest molecules, and the interaction is specifically represented by: the guest acts with the metal ion coordination in the host and the weak bond interaction between the guest and the ligand in the host, and thus the ME and AIE effects can be simultaneously exhibited.
The invention also provides a preparation method of the composite ICPs, which comprises the steps of mixing the ligand and the guest in a buffer solution, and then adding the aqueous solution of metal ions into the buffer solution for stirring to obtain the composite ICPs.
Wherein the final concentration of ligand in the buffer solution comprising ligand and guest is 1-10 mM; preferably, it is 5 mM.
Wherein the final concentration of the guest in the buffer solution containing the ligand and the guest is 0.01-1 mg/mL; preferably, it is 0.1 mg/mL.
Wherein the final concentration of the aqueous solution of metal ions is 1-10 mM; preferably, it is 5 mM.
Wherein the concentration of the buffer solution is 0.01M-1M; preferably, it is 0.1M.
Wherein the pH of the buffer solution is 3-10; preferably, the pH is 7.4.
Wherein the buffer solution is a tris (hydroxymethyl) aminomethane, a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution and the like; preferably, the buffer solution is 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution.
Specifically, the buffer solution is a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the concentration of 0.1M and the pH value of 7.4.
Wherein the stirring temperature is 20-40 ℃; preferably, it is 25 ℃.
Wherein the stirring time is 3min-15 min; preferably, it is 5 min.
The preparation method of the composite ICPs also comprises the step of washing and centrifuging the stirred system for a plurality of times to discard supernatant.
In one embodiment, the composite ICPs are prepared according to the following steps: and adding the water solution of the central metal ions into the buffer solution containing the ligand and the guest, stirring for 5min at room temperature, centrifuging, and washing to obtain the composite ICPs (water-soluble tetra (4-sulfophenyl) ethylene sodium salt guest functionalized ICPs nanoparticles).
The technical difficulty of the invention is that the order of adding the guest, the ligand and the metal ions is as follows: firstly, an object and a ligand need to be uniformly mixed, and the ligand and the non-covalent part of the object limit the rotation of a benzene ring in the object, so that the object shows the ME characteristic; subsequently, metal ions are added, and composite ICPs are formed at this time due to coordination of the metal ions with the guest, so that they exhibit AIE characteristics.
Therefore, in the preparation method of the composite ICPs, the ligand and the guest must be mixed to sensitize guest ME, and then metal ions are added to form the composite ICPs, so that ME and AIE can be regulated and controlled simultaneously.
The invention also provides the aggregation-induced fluorescence guest functionalized composite infinite coordination polymer nano particle prepared by the method.
The invention also provides a novel visual sensing mechanism for regulating and controlling multiple responses of the aggregation-induced fluorescent guest functionalized composite infinite coordination polymer nanoparticles based on metal competitive coordination, the optical characteristics of the central metal ions of the host can be changed by utilizing the characteristic that a substance to be detected has stronger coordination with the central metal ions of ICPs, and simultaneously, the appearance of the composite ICPs is changed by the coordination of the substance to be detected and the central metal ions, so that in the process of converting the guest from an aggregation state to a dispersion state, AIE is reduced, ME is increased, and various novel visual sensing mechanisms are established by utilizing the multiple responses of the spectrum and the obvious change of fluorescence color caused by the multiple responses.
Specifically, the coordination mode of the compound ICPs is destroyed by utilizing the strong coordination of a substance to be detected, such as 2, 6-dipicolinic acid (DPA), and europium ions in the center of the ICPs, so that the framework structure of a host network of the compound ICPs is broken, the water-soluble tetraphenylethylene of the object is converted from an aggregation state to a free state, the fluorescence emission (AIE, 455nm) of the object in the aggregation state is weakened, and the fluorescence emission (ME, 390nm) of the object in a monomer state is strengthened; meanwhile, the substance to be measured (DPA) transfers its own energy to the central metal europium ion through coordination (antenna effect), and the fluorescence is sensitized (615 nm). By regulating and controlling the metal competitive coordination effect, the spatial conformation of a host is changed by the stimulus response of WSSu-TPE @ Eu/GMP composite ICPs (water-soluble tetra (4-sulfophenyl) ethylene sodium salt object functionalized ICPs nano particles) to a substance to be Detected (DPA), so that the multiple optical response and the obvious change of fluorescence color of the host and the object are caused, and a brand new visual sensing mechanism is established.
The invention also provides application of the aggregation-induced fluorescent guest functionalized composite infinite coordination polymer nano particle in visual detection of 2, 6-dipicolinic acid based on metal competitive coordination regulation and control.
The invention also provides a novel method for double-proportion fluorescence visualization analysis of 2, 6-dipicolinic acid, which comprises the following specific steps:
the 2, 6-pyridinedicarboxylic acid standard is added to the buffer dispersion containing the composite ICPs, and after a reaction time, the measurement is carried out by a fluorimeter under a certain excitation wavelength. And respectively drawing standard curves 1 and 2 by taking the concentration of DPA as an abscissa and the ratio of fluorescence response values at 390nm and 455nm and the ratio of fluorescence response values at 615nm and 455nm (or 592nm and 455nm, 652nm and 455nm, 693nm and 455nm) as an ordinate to construct a double-proportion fluorescence visualization analysis method. Simultaneously, under the excitation of an ultraviolet lamp, a photo is taken in a darkroom, and the R value and the B value are recorded by using color analysis software. And (3) drawing a standard curve 3 by taking the concentration of the DPA as an abscissa and the ratio of the R value to the B value in the picture as an ordinate, and establishing a direct visual analysis method.
The double-proportion fluorescence visualization analysis method is characterized in that detection information can be expressed by the ratio of signal intensity changes at different fluorescence wavelengths, and a target object is usually selected as the target objectTwo peaks, one rising and one falling, were used. 390nm and 455nm belong to ME peak and AIE peak of WSSu-TPE respectively, so F is selected as standard curve 1390/F455As a ordinate; but because Eu3+Four peaks are 592nm, 615nm, 652nm and 693nm after sensitization, so that F is removed by the standard curve 2615/F455Optionally F592/F455、F652/F455、F693/F455As an ordinate. If the metal ions and the ligands in the object or the host are replaced, the standard curve needs to be prepared again.
The invention establishes a double-proportion fluorescence visualization analysis method, aims to improve the accuracy and reliability of an analysis result in a complex environment system and overcome the problem of false positive interference.
The invention establishes a direct visualization analysis method, and aims to process a picture of an analysis result through a smart phone with image color recognition software, so that the problem that the visualization site accurate quantitative analysis is restricted by a large optical instrument is solved.
The reaction temperature in the invention is 20-40 ℃; preferably, it is 25 ℃.
The reaction time in the invention is 5s-30 s; preferably, it is 15 s.
The excitation wavelength is 260nm-300 nm; preferably 275 nm.
The invention preferably adopts the smart phone to take the photos.
The time for shooting the analyzed sample by adopting the smart phone is 3-7 min; preferably, it is 5 min.
In the buffer dispersion liquid of the composite ICPs, the concentration of the composite ICPs is 0.3235mg/mL-1.294 mg/mL; preferably, it is 0.647 mg/mL.
In the buffer dispersion of the composite ICPs of the present invention, the buffer dispersion is a tris (hydroxymethyl) aminomethane hydrochloric acid solution having a concentration of 10mM and a pH of 3 to 10, specifically, a tris (hydroxymethyl) aminomethane hydrochloric acid solution having a concentration of 10mM and a pH of 7.4.
The total volume of the buffer dispersion containing the composite ICPs was 1.0 mL.
The volume of the 2, 6-dipicolinic acid standard substance is 1-100 mu L; preferably, it is 10. mu.L.
Specifically, the method for analyzing the 2, 6-dipicolinic acid by adopting the double-proportion fluorescence visualization comprises the following steps:
(1) establishing a standard curve: carrying out quantitative analysis on the 2, 6-dipicolinic acid standard substance, taking the concentration of the 2, 6-dipicolinic acid as an abscissa, and respectively utilizing the ratio F of the monomer fluorescence emission intensity (ME, 390nm) and the aggregate fluorescence emission intensity (AIE, 455nm) of the guest WSSu-TPE390/F455Eu sensitized by DPA3+The ratio F of fluorescence emission to aggregate fluorescence emission intensity (AIE, 455nm)615/F455(or F)592/F455、F652/F455、F693/F455) And respectively drawing standard curves 1 and 2 for vertical coordinates, and establishing a double-proportion fluorescence visualization analysis method.
(2) Under the excitation of a 254nm ultraviolet lamp, a photo of the mixed solution is recorded in a dark room by using a smart phone, the concentration of the 2, 6-dipicolinic acid is taken as a horizontal coordinate, and the ratio of the R/B value in the photo is taken as a vertical coordinate, so that a direct visual chemical analysis method is established.
The invention also provides a novel multichannel response mode (method) for regulating and controlling the coffee ring deposition morphology based on composite ICPs stimulus response, which specifically comprises the following steps: on a hydrophilic or hydrophobic substrate, the competitive action of the chromatographic separation effect of the coffee ring under the action of capillary driving force in the horizontal direction and the evaporation effect in the vertical direction is regulated, the particle size and the number in the stimulation response process of the composite ICPs are changed at the same time, and finally the particle size and the number are reflected by different coffee ring deposition appearances as a new signal reading mode and are combined with fluorescence, color and the like to establish a new multichannel response mode (method). By combining the unique AIE effect of the object, a multichannel visual test paper is developed to realize sensitive and reliable analysis of molecules to be detected (such as DPA).
The hydrophilic substrate specifically refers to Whatman1# test paper and other qualitative and quantitative filter paper with similar performance and an Aminopropyltriethoxysilane (APS) treated glass chip.
Wherein the hydrophobic substrate is a glass chip treated by hexadecyl trimethoxy silane (HDS).
Wherein the different coffee ring deposition profiles comprise: spotting (coffee ring inhibition), ring formation (coffee ring generation), different area of the spot from ring, different ring diameter of the spot from ring or other differences in morphology.
The invention also provides application of the multichannel response mode (method) for regulating and controlling the coffee ring deposition morphology by the stimulation response of the composite ICPs in the determination of 2, 6-dipicolinic acid in the bacillus subtilis spores.
The invention also provides a coffee ring product, which is a multi-channel response product for regulating and controlling the deposition morphology of the coffee ring on different substrates based on the stimulation response of the composite ICPs, and specifically comprises the following components: on various qualitative and quantitative filter paper test paper or various glass chip substrates, by adjusting the conditions of temperature, the aperture of the test paper substrate, the hydrophilicity and hydrophobicity of the substrate and the like, the competitive action of the chromatographic separation effect of the coffee ring and other effects (such as filtration effect, evaporation effect, Marangoni effect and the like) is utilized, the particle size and the number in the stimulation response process of the composite ICPs nano particles are changed at the same time, the particle size and the number are reflected by different coffee ring deposition appearances (such as spot formation (inhibition), ring formation, the area size of a spot ring and the ring diameter size), the detection method is expanded into a new signal reading mode, and the coffee ring product which can be used for accurate quantitative analysis is developed by combining the changes of color, fluorescence and the like.
The coffee ring product comprises coffee ring test paper, a coffee ring chip and the like.
The invention also provides application of the coffee ring product in detecting 2, 6-dipicolinic acid.
The invention also provides a preparation method of the coffee ring product, which comprises the following steps:
adding a series of 2, 6-pyridinedicarboxylic acid standard samples with different concentrations into the buffer dispersion liquid of the composite ICPs to prepare a mixture, dripping a certain volume of the mixture on a substrate, and waiting for the mixture to dry under the condition of certain temperature and humidity to prepare a multi-channel response type coffee ring product.
In the buffer dispersion liquid of the composite ICPs, the concentration of the composite ICPs is 1.941mg/mL-3.882 mg/mL; preferably, it is 3.24 mg/mL.
In the buffer dispersion of the composite ICPs of the present invention, the buffer dispersion is a tris (hydroxymethyl) aminomethane hydrochloric acid solution having a concentration of 10mM and a pH of 3 to 10, specifically, a tris (hydroxymethyl) aminomethane hydrochloric acid solution having a concentration of 10mM and a pH of 7.4.
The total volume of the buffer dispersion containing the composite ICPs was 1.0 mL.
The volume of the 2, 6-dipicolinic acid standard substance is 1-100 mu L; preferably, it is 10. mu.L.
The substrate is various qualitative and quantitative filter paper test paper or various glass chips.
The qualitative and quantitative filter paper test paper is Whatman1# test paper.
The certain volume specifically means 5 μ L.
The certain temperature and humidity are specifically 25 ℃ and the relative humidity is 46%.
The drying of the invention is natural drying.
The invention also provides a coffee ring test paper, the substrate of which is hydrophilic test paper, the coffee ring test paper comprises Whatman1# test paper and other qualitative and quantitative filter paper with similar performance, and the diameter of the filter paper is 5-10 mm; preferably 6 mm.
The coffee ring test paper of the invention can present different deposition appearances, including: spotting (coffee ring inhibition), ring formation (coffee ring generation), different area of the spot from ring, different ring diameter of the spot from ring or other differences in morphology.
The multiple response mode of the coffee ring test paper can be used as self-contrast among different channels, so that the problem of single signal reading mode in the existing test paper and the prior art is solved, and the accuracy and the reliability of an analysis result in a complex environment system are improved.
The invention also provides application of the coffee ring test paper in detection of 2, 6-dipicolinic acid.
The invention also provides a method for detecting 2, 6-dipicolinic acid by using the multichannel coffee ring test paper, which comprises the steps of adding a series of 2, 6-dipicolinic acid standard substances with different concentrations into the buffer dispersion liquid of the composite ICPs, uniformly mixing, dripping the mixture on a test paper substrate, shooting an analysis result picture in a dark room under the excitation of an ultraviolet lamp, and constructing the multichannel coffee ring test paper according to corresponding changes of colors of test paper spot ring patterns and central spots of the 2, 6-dipicolinic acid standard substances with different concentrations, so as to realize quantitative analysis on the 2, 6-dipicolinic acid standard substances.
Specifically, the method for detecting 2, 6-dipicolinic acid by using the multichannel coffee ring test paper comprises the following steps:
a series of 2, 6-pyridinedicarboxylic acid standards with different concentrations are added into the buffer dispersion liquid of the composite ICPs, and after being mixed uniformly, the mixture is dripped on a hydrophilic test paper. Under the excitation of a 254nm ultraviolet lamp, a picture of an analysis result is shot in a darkroom by using a smart phone, the R value and the B value of the center of a test paper spot are recorded by using color analysis software, the ratio of the R/B value is taken as a vertical coordinate, the DPA concentration is taken as a horizontal coordinate, and a standard curve 4 (channel 1) is drawn. Meanwhile, analyzing the halo area A covered by Eu/DPA small molecules by using image analysis softwareringLight spot area A covered with composite ICPs materialspotWith Aring/AspotThe ordinate is the ordinate and the DPA concentration is the abscissa, and a standard curve 5 (channel 2) is plotted.
The invention aims to draw the standard curves 4 and 5 to realize the determination of the concentration of the 2, 6-dipicolinic acid in a sample by using different channels of the test paper, and the detection results among the channels can be subjected to self-contrast, thereby improving the reliability of the test paper.
The concentration of the compound ICPs is 3.24mg/mL
The volume of the mixture of 2, 6-dipicolinic acid standard and composite ICPs was 5. mu.L.
The invention also provides a visualization method directly used for the field analysis of 2, 6-dipicolinic acid in bacterial spores, which specifically comprises the following steps:
replacing the 2, 6-dipicolinic acid standard substance in the novel double-proportion fluorescence visualization analysis method for 2, 6-dipicolinic acid with bacterial spore suspension, measuring the ratio of corresponding fluorescence response values of the actual sample according to the novel double-proportion fluorescence visualization analysis method for 2, 6-dipicolinic acid, substituting the ratio into a standard curve 1 and a standard curve 2, and measuring the concentration of the actual sample 2, 6-dipicolinic acid to obtain results 1 and 2. According to the new double-proportion fluorescence visualization analysis method for the 2, 6-dipicolinic acid, the corresponding R/B ratio of the actual sample is measured by combining a smart phone, the R/B ratio is substituted into the standard curve 3, the concentration of the actual sample 2, 6-dipicolinic acid is measured, a result 3 is obtained, and then self-comparison is carried out with the results 1 and 2.
Replacing the 2, 6-dipicolinic acid standard substance in the coffee ring test paper for the 2, 6-dipicolinic acid with bacterial spore suspension, measuring the ratio of the center R/B of the light spot on the test paper by combining a smart phone, and substituting the ratio into a standard curve 4 to obtain a result 4; on the analytical test paper Aring/AspotSubstituting the standard curve 5 to obtain a result 5; and (4) carrying out self-control on the obtained results 4 and 5 to ensure the accuracy of the analysis result.
The bacterial spore suspension, in particular to the spore suspension prepared by treating the bacillus subtilis with alanine solution at 70 ℃ for 30 minutes and 60 minutes and filtering to remove thalli, is not limited to the bacteria.
The compound ICPs can be used for realizing the practical application of the field analysis of the 2, 6-dipicolinic acid in the bacterial spore suspension.
The multi-channel coffee ring test paper can be used for realizing the practical application of on-site analysis on the 2, 6-dipicolinic acid in the bacterial spore suspension.
Compared with the prior art, the invention has the following advantages:
1) according to the invention, an aggregation-induced emission material is innovatively selected as an object, and a composite infinite coordination polymer nanoparticle with ME and AIE effects is prepared by regulating the interaction between the object and the object, so that the requirement on the optical performance of a host material is lowered, and the possibility is provided for developing a new visual sensing mechanism design based on stimulus-responsive composite ICPs;
2) according to the invention, through designing a metal competitive coordination effect, the host conformation is changed by utilizing the stimulus response of the composite ICPs to DPA, and the ME and AIE effects of an object and the sensitization effect of metal ions in the center of the host are regulated and controlled at the same time, so that a brand new visual sensing mechanism of stimulus response type composite ICPs is established, and the problem of single sensing mechanism is solved; a double-proportion fluorescence visualization analysis method is developed, and the accuracy and reliability of DPA field analysis results are effectively improved;
3) the invention creatively combines the stimulation response of the composite ICPs with the coffee ring effect, regulates the morphology of coffee ring deposition through the change of the size and the number before and after the stimulation response of the composite ICPs nano particles, converts the morphology into a new signal reading mode, and constructs a new multi-channel sensing mode by combining the change of fluorescence color. On the basis, the unique optical advantages of the object are fully utilized to develop the multi-channel coffee ring test paper, the analysis result of each channel can be used as self contrast, and the reliability of the test paper in the actual quantitative analysis application is ensured.
4) The invention processes the picture of the analysis result through the intelligent mobile phone with image recognition and data processing software, really solves the problem of relying on a large-scale optical instrument in visual analysis, and can meet the requirement of on-site rapid detection of a large batch of samples in an emergent public safety event.
Drawings
FIG. 1 is an SEM photograph, SEM-EDS (inset) (A) and fluorescence spectrum (B) of WSSu-TPE @ Eu/GMPECPs.
FIG. 2 shows the working principle of the WSSu-TPE @ Eu/GMPECPs prepared by the present invention for detecting DPA.
FIG. 3 is SEM image (A) and fluorescence spectrum (B) of WSSu-TPE @ Eu/GMPECPs after DPA action.
FIG. 4 is a graph showing the two-ratio type fluorescence response curve (A) and linearity (B, C) of WSSu-TPE @ Eu/GMP ICPs under the action of DPA.
FIG. 5 is a photograph of the change in fluorescence color of WSSu-TPE @ Eu/GMPECPs with DPA (A) and a line graph based on smartphone analysis (B).
FIG. 6 is a diagram of the working principle of the multi-channel responsive coffee ring test paper for DPA detection.
FIG. 7 is a graph of the deposition pattern (A) and the linearity (B, C) of a multi-channel responsive coffee ring test strip.
Fig. 8 is a smartphone-based visual field analysis of DPA of bacillus subtilis released spores with a multi-channel coffee ring test paper.
Fig. 9 is a real-time monitoring of bacillus subtilis release of DPA using confocal laser microscopy.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Example 1 preparation and characterization of aggregation-induced fluorescent monomer functionalized composite infinite coordination polymer nanoparticles simultaneously exhibiting ME and AIE response properties
WSSu-TPE was prepared according to the literature (New J. chem.2017,41, 4747-4749; Supermol. chem.2017,30,1-8), with tetraphenylethylene (1.00g, 3.0mM) dispersed in 20mL of concentrated sulfuric acid and heated at 120 ℃ for 3 hours. Thereafter, 10mL of water and 30mL of ethyl acetate were added to the mixture to terminate the reaction. The resulting precipitate was filtered and washed twice with 10mL ethyl acetate to give pure TPE-SO3H, grey solid. Then, TPE-SO3The H was dissolved in 20mL of water and neutralized with 0.1M aqueous NaOH. After freeze-drying, the solid obtained was washed twice with 10mL of acetone to obtain TPE-SO as a yellow solid3Na is WSSu-TPE.
5mL of an aqueous solution of 10mM europium nitrate was added with stirring to 5mL of a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution (0.1M, pH 7.4) containing 10mM guanosine-5' -monophosphate and 0.2mg/mL of WSSu-TPE. Wherein the final concentration of europium nitrate is 5mM, the final concentration of guanosine-5' -monophosphate is 5mM, and the final concentration of WSSu-TPE is 0.1 mg/mL. Stirring at room temperature for 5min, centrifuging, washing with 2mL deionized water for 3 times to obtain compound ICPs (WSSu-TPE @ Eu/GMP). FIG. 1A is an SEM image of WSSu-TPE @ Eu/GMP, from which it can be seen that WSSu-TPE @ Eu/GMP is a regular spherical nanoparticle, the appearance of the S element (from WSSu-TPE) in SEM-EDS (inset) also indicates that the guest is successfully encapsulated in the host network structure, FIG. 1B is a fluorescence spectrum of WSSu-TPE @ Eu/GMP ICPs, which indicates that the ME of WSSu-TPE is sensitized when the ligand is present, and that WSSu-TPE is converted from a dispersed state to an aggregated state after WSSu-TPE @ Eu/GMP is formed by regulation of host-guest action, the sensitized ME (390nm) is attenuated, and the AIE (455nm) is enhanced.
Example 2 DPA double proportion type fluorescence visualization analysis method based on WSSu-TPE @ Eu/GMPECPs
The principle of the double-proportion fluorescence visualization analysis method of the invention is shown in FIG. 2, and utilizes the extremely strong coordination (logK) between DPA and EuEu-DPA7.46 ± 0.02) ligands GMP and Eu are disrupted3+Coordination of (logK)Eu-GMP4.04 +/-0.02), and the main body structure is damaged, on one hand, WSSu-TPE is converted from an aggregation state to a dispersion state, ME (390nm) is enhanced, AIE (455nm) is weakened, and on the other hand, DPA and Eu are reduced3+Will transfer self energy to Eu after coordination3+Sensitize its fluorescence (615 nm). FIG. 3A is an SEM image of the WSSu-TPE @ Eu/GMP ICPs prepared in example 1 of the present invention after being reacted with DPA, which shows that the host structure of the composite ICPs is indeed completely destroyed in the presence of DPA. Meanwhile, as shown in fig. 3B, when DPA exists, the fluorescence spectrum of WSSu-TPE @ Eu/gmpcs shows triple optical responses of the central metal ion and the object, and the triple optical responses have no spectrum cross interference, and the fluorescence color shows obvious change from blue to red, thereby laying a foundation for constructing a double-scale fluorescence visualization method.
mu.L of a DPA standard sample at a series of concentrations (0. mu.M, 8. mu.M, 10. mu.M, 50. mu.M, 0.1mM, 0.3mM, 0.5mM, 0.7mM, 1mM, 1.5mM, 2mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM in this order) was added to 990. mu.L of HEPES buffer (10 mM, pH 7.4) containing 0.647mg/mL of WSSu-TPE @ Eu/GMP prepared in example 1 of the present invention (final concentrations are 0, 0.08, 0.10, 0.50, 1.0, 3.0, 5.0, 7.0, 10, 15, 20, 25, 30, 35, 40. mu.M in this order) for 15 seconds, and then the measurement was performed using a fluorometer (lambda. mu.M)ex275nm) and the resulting fluorescence spectrum is shown in fig. 4A. Taking DPA concentration as abscissa, respectively using ratio of fluorescence response values at 390nm and 455nm, and fluorescence response values at 615nm and 455nmThe ratio of the fluorescence response values is plotted on the ordinate and the standard curves 1, 2 can be plotted as shown in FIG. 4B, C. As shown in fig. 4B, the unary linear equation corresponding to the standard curve 1 is a ═ 0.084X +0.217 (R)20.995), A is F390/F455X is DPA concentration in μ M with a minimum detection limit of 30 nM. As shown in FIG. 4C, the standard curve 2 corresponds to a one-dimensional linear equation of 0.247X +0.099 (R)20.996), A is F615/F455No unit, (or a ═ 0.012x +0.355 (R)20.978), a is F592/F455No unit; a is 0.0055x-0.0006 (R)20.970), a is F652/F455No unit; a ═ 0.039x-0.046 (R)20.983), A is F693/F455And no unit. ) X is the concentration of DPA in μ M with a minimum detection limit of 27 nM. The above mixed solution was photographed in a dark room using a smartphone under excitation of a 254nm uv lamp (see fig. 5A), and R and B values were recorded using color analysis software. A standard curve 3 is drawn by using the DPA concentration as an abscissa and the ratio of the R value to the B value in the picture as an ordinate, and as shown in fig. 5B, the unary linear equation is that a is 0.025X +0.167(R is ═ B (R ═ B { (R {)20.992), a is R/B, nothing, X is the concentration of DPA in μ M, the lowest concentration detected is 100 nM.
Example 3 multichannel coffee Ring test strips based on the response of WSSu-TPE @ Eu/GMPECPs to DPA stimulation
mu.L of a series of concentrations (500. mu.M, 1mM, 5mM, 10mM, 15mM, 20mM in this order) of 2, 6-pyridinedicarboxylic acid standard samples were added to 990. mu.L of 3.24mg/mL of the buffered dispersion of composite ICPs prepared in example 1 of the present invention to prepare a mixture (final concentration of 5, 10, 50, 100, 150, 200. mu.M). 5 μ L of the mixture was dropped onto Whatman # 1 hydrophilic test strips of about 6mm diameter, and dried at 25 ℃ and a relative humidity of 46% to produce multichannel responsive coffee ring strips.
As shown in FIG. 6, the principle underlying the present invention is that the capillary driving force-based coffee ring chromatographic separation effect competes with the vertical evaporation effect in the analysis zone fixed on the test paper, and when the concentration of 2, 6-pyridinedicarboxylic acid is different, the pattern of the coffee ring formed by WSSu-TPE @ Eu/GMP is also different. The coffee ring effect of the capillary driving force can be counteracted by the larger size of WSSu-TPE @ Eu/GMP evaporation, thereby depositing a spot coverage test paper. At the same time, the spot exhibited a bright blue fluorescent color under excitation of a 254nm UV lamp due to the AIE effect of the guest WSSu-TPE. However, with the addition of DPA, the structure of the WSSu-TPE @ Eu/GMP composite ICPs material is destroyed, and an Eu/DPA composite with small size and strong red fluorescence emission is formed, so that a ring structure with red fluorescence appears on the periphery of the test paper. With the further increase of the concentration of the 2, 6-pyridinedicarboxylic acid, the capillary flow can simultaneously adjust the change of the particle size and the amount of the composite ICPs material, so that the fluorescence color of the spots gradually changes from blue to red, and simultaneously the area of the peripheral ring becomes larger and the area of the central spot becomes smaller.
Using image recognition and processing software installed in a smartphone to analyze a manufactured multi-channel responsive coffee ring test paper (fig. 7A), taking the concentration of DPA as an abscissa, respectively taking the ratio of the R value of the coffee ring test paper annular pattern to the B value of the central speckle pattern, and the ratio of the area of the coffee ring test paper annular pattern to the area of the central speckle pattern as an ordinate, drawing standard curves 4 and 5, as shown in fig. 7B, where a unary linear equation corresponding to the standard curve 4 is that a is 0.0054X +0.171(R is 0.0054X + 0.171) (R is a linear equation)20.991), wherein a is the ratio of R/B values at the central spot of the coffee ring test paper, without unit, X is the concentration of DPA in μ M, and the lowest detected concentration is 5 μ M; as shown in fig. 7C, the unary linear equation corresponding to the standard curve 5 is a ═ 0.0296X +0.138 (R)20.990), wherein a is the area of the ring spot aring/AspotX is DPA concentration in. mu.M, and the lowest concentration detected is 5. mu.M.
Example 4 DPA in-situ analysis based on double-proportion fluorescence visualization method and multichannel coffee ring test paper
The spore suspension was prepared as follows: bacillus subtilis was inoculated on Luria-Bertani medium (10g/L of trypsin, 5g/L of yeast extract, 10g/L of NaCl), cultured in a constant temperature shaking incubator (150r/min) at 37 ℃ for 72 hours, centrifuged and washed several times until the supernatant was colorless, and then collected, freeze-dried, and stored in a refrigerator at 4 ℃.10 mg of Bacillus subtilis powder is dissolved in 100 mu L of alanine solution with the concentration of 10mM, and the solution is respectively subjected to water bath for a period of time at the temperature of 70 ℃, and spore suspension is prepared after thalli are removed by filtration.
The 2, 6-dipicolinic acid standard in example 2 of the present invention was replaced with the spore suspension, the spore suspensions in the water bath were measured for 30 minutes and 60 minutes as described in example 2, and the results are shown in FIG. 8A by substituting the fluorescence responses of the spore suspensions into the standard curves 1, 2, and 3, respectively. The concentrations of DPA measured in Standard Curve 1 after alanine treatment for 30min and 60min were 22.56. + -. 0.03. mu.M, respectively; 36.07. + -. 08.06. mu.M, the concentrations of DPA measured in Standard Curve 2 were 22.53. + -. 0.02. mu.M and 36.09. + -. 0.07. mu.M, respectively, and the concentrations of DPA measured in Standard Curve 3 were 23.94. + -. 0.26. mu.M and 34.13. + -. 0.15. mu.M, respectively.
The 2, 6-dipicolinic acid standard in example 3 of the present invention was replaced with the spore suspension, and the coffee ring pattern obtained was analyzed according to example 3 and substituted into the standard curves 4 and 5 to obtain the DPA concentration in the spore suspension, as shown in fig. 8B. After alanine treatment for 30 minutes and 60 minutes, the concentrations of DPA measured in the standard curve 4 of the test paper are 26.04 +/-0.31 mu M and 37.05 +/-0.24 mu M respectively; the concentrations of DPA measured in the test paper standard curve 5 are respectively 25.19 +/-0.46 mu M; 38.41. + -. 0.35. mu.M. The DPA concentrations obtained by different channels of the multi-channel response type coffee ring test paper are high in coincidence degree, in addition, the analysis results of the multi-channel response type coffee ring test paper have better consistency regardless of a double-proportion type visualization method or a multi-channel test paper, the reliability of the multi-channel response type coffee ring test paper in the analysis of complex samples is essentially ensured, and the multi-channel response type coffee ring test paper can be directly used for the field analysis of the DPA in actual samples.
Example 5 real-time monitoring of Bacillus subtilis Release of DPA
The spore suspension of the invention in example 4 was used and mixed with the composite ICPs of the invention prepared in example 1 after water bath for 0min, 30min and 60 min. Subsequently, images of the blue channel (430-500nm) and the red channel (580-650nm) were collected by confocal laser microscopy, as shown in FIG. 9. As the water bath time increased, the image showed a clear change: fluorescence color change of blue channelWeak, the luminescence of the red channel becomes stronger. This phenomenon is due to the fact that the complex ICPs can be electrostatically adsorbed on the spores and then react with DPA released from the spores. As the spore release DPA increases, the destruction of complex ICPs results in a decrease in blue channel fluorescence intensity; released DPA and Eu3+The reaction produces a Eu/DPA complex, resulting in an increase in the fluorescence of the red channel. Therefore, double-response optical change around the spores can be observed, and real-time monitoring of DPA released by the bacillus subtilis is realized.
In conclusion, WSSu-TPE with aggregation-induced emission based performance is innovatively selected as an object, the interaction between the object and the object is reasonably designed, and the WSSu-TPE @ Eu/GMPECPs nano material with ME and AIE effects is prepared. By designing competitive coordination among DPA, ligand and central metal ions, the spatial conformation of a main body is further destroyed, Monomer Emission (ME) and aggregate fluorescence emission (AIE) of WSSu-TPE are regulated and controlled, and Eu is sensitized simultaneously3+Fluorescence, a new visual sensing mechanism is constructed by utilizing the triple optical response, and a more reliable new double-proportion fluorescence visual analysis method is developed; meanwhile, the multi-channel response type coffee ring test paper is invented based on the stimulation response regulation and control of the coffee ring deposition morphology of the composite ICPs material nano particles, has the advantages of low cost, convenience in carrying, quick response time, small required sample amount, simplicity in operation, high analysis result goodness of fit and the like, and can directly realize simple and reliable quantitative detection of DPA in a sample to be detected. The method can be directly used for the field analysis of DPA in the bacillus subtilis spores, and has a larger practical application prospect in the related fields of social public safety.
The above embodiments are only for illustrating the technical concept and features of the present invention, so that those skilled in the art can understand the contents of the present invention and implement the present invention, and the protection scope of the present invention is not limited thereby. All equivalent changes and modifications made in accordance with the spirit of the present disclosure are intended to be included within the scope of the present disclosure.
Claims (14)
1. A composite infinite coordination polymer nanoparticle composite ICPs with fluorescence guest functionalization induced by aggregation is characterized by specifically comprising a host and a guest; the main body is formed by freely coordinating and combining metal ions and corresponding ligands;
wherein the metal ion is a lanthanide metal ion including europium ion (Eu) as a central ion3+) Terbium ion (Tb)3+) Cerium ion (Ce)3+) Lanthanum ion (La)3+) Neodymium ion (Nd)3+) Gadolinium (Gd)3+) Erbium ion (Er)3+) Ytterbium ion (Yb)3+) One or more of;
the ligand is an alkaloid ligand, and comprises one or more of guanosine-5 '-monophosphate (GMP), adenosine-5' -monophosphate (AMP), uridine-5 '-monophosphate (UMP), cytidine-5' -monophosphate (CMP), guanosine-5 '-diphosphate (GDP), adenosine-5' -diphosphate (ADP), uridine-5 '-diphosphate (UDP), cytidine-5' -diphosphate (CDP), guanosine-5 '-triphosphate (GTP), adenosine-5' -triphosphate (ATP), uridine-5 '-triphosphate (UTP), cytidine-5' -triphosphate (CTP);
the object is tetra (4-sulfophenyl) ethylene sodium salt;
the molar ratio of the metal ions, the ligand and the guest is 37:37 (1-10).
2. The preparation method of the aggregation-induced fluorescence guest functionalized composite infinite coordination polymer nanoparticle according to claim 1, characterized in that the ligand and the guest are mixed in a buffer solution, and then an aqueous solution of metal ions is added and stirred to obtain the composite infinite coordination polymer nanoparticle.
3. The method according to claim 2, wherein the final concentration of the ligand in the buffer solution comprising the ligand and the guest is 1 to 10 mM; and/or the final concentration of the guest molecule in the buffer solution containing the ligand and the guest is 0.01-1 mg/mL; and/or the final concentration of the aqueous solution of the metal ions is 1-10 mM.
4. The aggregation-induced fluorescence guest functionalized composite infinite coordination polymer nanoparticle prepared by the method according to claim 2 or 3, which is characterized in that the nanoparticle can simultaneously exhibit the effects of monomer emission ME and aggregate emission AIE under the regulation and control of the host and the guest.
5. The use of the aggregation-induced fluorescent guest-functionalized complex infinite coordination polymer nanoparticle according to claim 1 or 4 for the visual detection of 2, 6-pyridinedicarboxylic acid.
6. A method for double-proportion type fluorescence visualization analysis of 2, 6-dipicolinic acid, characterized in that a 2, 6-dipicolinic acid standard is added to a buffer dispersion containing the composite ICPs as defined in claim 1, after a reaction period, measurement is performed by a fluorometer at a certain excitation wavelength, standard curves 1 and 2 are respectively drawn with the concentration of DPA as abscissa, the ratio of fluorescence response values at 390nm and 455nm, respectively, and the ratio of fluorescence response values at 615nm and 455nm, or 592nm and 455nm, or 652nm and 455nm, or 693nm and 455nm, respectively, as ordinate, to construct a double-proportion type fluorescence visualization analysis method; meanwhile, a photo is shot in a darkroom under the excitation of an ultraviolet lamp, a color analysis software is used for recording an R value and a B value, the concentration of DPA is taken as an abscissa, the ratio of the R value to the B value in the photo is taken as an ordinate, a standard curve 3 is drawn, and a direct visual analysis method is established.
7. The method of claim 6, wherein the temperature of the reaction is 20 ℃ to 40 ℃; and/or the reaction time is 5s-30 s; and/or the excitation wavelength is 260nm-300 nm; and/or, in the buffer dispersion liquid containing the composite ICPs, the concentration of the composite ICPs is 0.3235mg/mL-1.294 mg/mL; and/or the volume of the 2, 6-dipicolinic acid standard is 1-100 mu L.
8. A multi-channel response method for regulating coffee ring deposition profiles based on the stimulation responses of composite ICPs as claimed in claim 1, wherein on a hydrophilic or hydrophobic substrate, the size and number of particles in the stimulation response process of composite ICPs are changed by regulating the competition effect of the coffee ring chromatographic separation effect under the action of capillary driving force in the horizontal direction and the evaporation effect in the vertical direction, and are reflected by different coffee ring deposition profiles as a new signal readout mode to be combined with fluorescence and color to establish a multi-channel response method.
9. A coffee ring product, wherein the coffee ring product is a multi-channel response product based on the composite ICPs stimulus response modulated coffee ring deposition profile of claim 1 on different substrates.
10. The coffee ring product of claim 9, wherein the coffee ring product is a coffee ring test paper, a coffee ring chip; the coffee ring test paper is characterized in that the substrate of the coffee ring test paper is hydrophilic test paper, and the coffee ring test paper comprises Whatman1# test paper and other qualitative and quantitative filter paper with similar performance, wherein the diameter of the filter paper is 5-10 mm.
11. The method of claim 9, wherein 2, 6-dipicolinic acid standards are added to the buffered dispersion of composite ICPs of claim 1 to form a mixture, and a volume of the mixture is then drop coated onto a substrate, and allowed to dry at a temperature and humidity to form a multi-channel responsive coffee ring product.
12. The method of claim 11, wherein the concentration of the complexed ICPs in the buffered dispersion of complexed ICPs is from 1.941mg/mL to 3.882 mg/mL; and/or the volume of the 2, 6-dipicolinic acid standard substance is 1-100 muL; and/or, the temperature and humidity specifically refer to 25 ℃ and a relative humidity of 46%.
13. The use of the multi-channel response method of modulating coffee ring deposition morphology in response to stimulation of composite ICPs according to claim 8 or the coffee ring product of claim 9 in the detection of 2, 6-pyridinedicarboxylic acid.
14. The method for detecting 2, 6-dipicolinic acid in a coffee ring product according to claim 9, wherein a standard sample of 2, 6-dipicolinic acid is added dropwise to the buffer dispersion of composite ICPs according to claim 1, and then coated on a test paper substrate, and a photo of the analysis result is taken in a dark room under the excitation of an ultraviolet lamp, and a multichannel coffee ring test paper is constructed according to the corresponding changes of the color of the test paper spot ring pattern and the central spot generated by the 2, 6-dipicolinic acid standard with different concentrations, so as to realize quantitative analysis of the 2, 6-dipicolinic acid standard.
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