1 Introduction
Molecularly imprinted technology (MIT) is a kind of preparation technology that can obtain a polymer that completely matches the binding site of a specific target molecule in space, and can specifically recognize the target molecule through the idea of preparation and elution
[1]. Since Pauling put forward the "template theory" in 1940, the design, preparation, characterization and application of molecularly imprinted polymers have been developed continuously
[2]. The preparation of Molecularly imprinted polymers (MIPs) by molecular imprinting technology generally includes three steps: first, target molecules and functional monomers form prepolymers by electrostatic/ionic, non-covalent, covalent, semi-covalent and metal-centered coordination interactions; Condly, a crosslinking agent and an initiator are added to initiate a polymerization reaction to form a highly crosslinked polymer around the previously synthesized prepolymer; Finally, the interaction between the target molecule and the functional monomer is disrupted, and the template molecule is eluted from the polymer (Figure 1), leaving a specific binding site that can selectively recognize the target molecule
[3].
In the process of constructing molecularly imprinted polymers, there are generally three polymerization mechanisms, the characteristics of which are shown in Table 1. (1) free radical polymerization. It is prepared by polymerizing vinyl functional monomers such as methacrylic acid (MAA), acrylamide (AAm) and 4-vinylpyridine (4-VP) with crosslinking agents such as ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM) and divinylbenzene (DVB). This method is widely used in the preparation of organic molecularly imprinted fluorescent sensors because of its simple experimental setup and easy availability of functional monomers. (2) Sol-gel method. Sol-gel silica was prepared mainly by condensation of tetraethoxysilane (TEOS) with functional monomers such as 3-aminopropyltriethoxysilane (APTES) under alkaline conditions. The molecularly imprinted polymer prepared by the method has good thermal stability and chemical stability, can well retain recognition sites, and is widely applied to the preparation of inorganic molecularly imprinted polymers. (3) Self-polymerization of dopamine. This method is a relatively novel method. Due to the hydrophilicity and biocompatibility of polydopamine (PDA), it is often used to prepare protein molecularly imprinted polymers through the self-polymerization of dopamine in weak alkaline aqueous solution. Cheng et al reviewed and analyzed the polymerization mechanism of dopamine forming PDA
[4]. Molecularly imprinted polymers (MIPs) have been widely used in the fields of sensors, sample pretreatment, degradation of environmental pollutants, drug analysis and cell imaging due to their specific recognition, thermal stability and wide range of applications
[5][14][15][16][17].
Molecularly imprinted polymers (MIPs) with good selectivity as the recognition element of sensors is an important application field of molecular imprinting technology, which has attracted wide attention of researchers in the field of analysis and detection. Traditional molecularly imprinted polymers (MIPs) lack the ability of signal transduction in practical applications, so many researchers have added fluorescent materials to MIPs to prepare molecularly imprinted fluorescent sensors. Molecularly imprinted fluorescent sensors based on molecularly imprinted polymers have been widely used in the detection of substances due to their excellent sensitivity.
When in contact with the target substance, the specific recognition site selectively binds to the target substance, and the fluorescence intensity changes accordingly, which is recorded by a fluorescence spectrophotometer or observed by naked eyes, thus realizing the selective recognition and fluorescence detection of the target substance
[18]. The prepared molecularly imprinted fluorescence sensor not only has good selectivity and stability of MIPs, but also has the advantage of high fluorescence detection sensitivity, does not need to use expensive instruments and complex sample pretreatment in practical application, can meet the rapid and high-sensitivity detection of complex samples, and is rapidly developed in sample detection
[19][20].
In this paper, the sensing mechanism of molecularly imprinted fluorescence (MIFS) and the types of fluorescent substances are mainly introduced, and the construction of different types of MIFS is emphatically introduced, including the design of single fluorophore detection of single target, ratiometric fluorescence detection of single target, and simultaneous detection of two targets. Finally, the current challenges and future prospects are proposed to accelerate the development of molecularly imprinted fluorescent sensors and expand their applications in environmental detection and adsorption.
2 Fluorophore Types and Fluorescence Sensing Principles
In the process of constructing molecularly imprinted fluorescent sensors, the selection of fluorophore materials can not be ignored. Common fluorescent materials include organic dyes, Metal nano clusters (MNCs), Quantum dots (QDs) and Carbon dots (CDs). Organic dyes have many advantages, such as variety, rich color, easy access and small size, but they have the disadvantages of narrow excitation peak, broad tail emission peak, poor stability and easy photobleaching, so their sensitivity is reduced and they are gradually replaced by other new fluorescent materials
[21]. Metal nanoclusters are generally composed of several to hundreds of metal atoms, and their size is close to the Fermi wavelength (< 2 nm), which is between a single metal atom and nanoparticles. Compared with larger nanoparticles, metal nanoclusters show excellent optical properties, such as tunable fluorescence emission, large Stokes shift, high fluorescence stability and high quantum yield
[22]. At present, various type of metal nanoclusters have been report, including gold nanoclusters (Au NCs), silver nanocluster (Ag NCs), copper nanoclusters (Cu NCs), platinum nanoclusters (Pt NCs) and molybdenum nanoclusters.Compared with traditional fluorescent dyes, the toxicity of metal nanoclusters is greatly reduced and the biocompatibility is significantly improved. As a new type of nano-fluorescent probe, metal nanoclusters have been widely used in the fields of biochemical analysis, environmental detection and medical imaging
[23][24][25][26][27][28,29]. In recent years, new fluorescent materials such as CDs and QDs have attracted much attention, among which QDs have the characteristics of high emission quantum yield, adjustable size, excellent photostability and high photoluminescence efficiency, while CDs have the characteristics of low toxicity, environmental friendliness and good water solubility, which will be more widely used in the field of efficient identification and determination of target molecules in the future
[30].
In that detection of the target analyte, the selective interaction between the fluorophore and the analyte can be express by quenching or signal enhancement of the fluorescence intensity. Changes in these signals are very sensitive to dose and allow quantitative detection of analyte concentrations. According to the principle of selective interaction between fluorophores and analytes, molecularly imprinted fluorescent chemosensors can be divided into three types: Fluorescence resonance energy transfer (FRET), Photoinduced electron transfer (PET) and Inner filter effect (IFE)
[31][32][33].
FRET is a pair of suitable fluorescent materials (energy donor and energy acceptor). In the range of 1.0 ~ 10 nm, when the emission spectrum of donor molecule overlaps with the absorption spectrum of acceptor molecule, the phenomenon of fluorescence energy transfer from donor to acceptor will occur. This process is a non-radiative energy transfer process, which is realized by the dipolar interaction between the donor and acceptor molecules
[34]. FRET requires that there must be an effective overlap between the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor, and that the distance between the energy donor and the energy acceptor must be small enough (1. 0 ~ 10 nm) to cause the transfer of fluorescence energy from the donor to the acceptor
[35].
PET refers to the electron transfer reaction of electron donor or acceptor induced by light excitation. Electron transfer sensors are mainly composed of three parts: fluorophor, linker arm and acceptor
[36]. When the target analyte is a quencher (acceptor), the target substance is in contact with the fluorophor, and the two are subjected to electron transfer so that the fluorophor is subjected to fluorescence quenching to form a specific fluorescent sensor with a connecting arm as a recognition element.When the target analyte is not present, the fluorophor and the acceptor are connected through the linker arm, and electron transfer occurs to cause the fluorescence of the fluorophor to recover, showing the "off" and "on" States of fluorescence
[37].
IFE refers to a decrease in fluorescence due to the absorption of excitation light or emission light by a phosphor or other light-absorbing substance when the phosphor is present in a large concentration or in the presence of other light-absorbing substances
[38]. IFE does not need to consider the distance between fluorophores and the functional groups of these fluorophores, and the direct overlap of the absorption spectrum of the absorber with the fluorescence spectrum of the fluorophore is the basis of the internal filtering effect
[39][40].
3 Construction of Molecularly Imprinted Fluorescent Sensor
Molecularly imprinted fluorescent sensors combine the advantages of fluorescent sensors, such as simplicity, rapidity, low cost, high sensitivity and high selectivity of molecular imprinting, and show obvious advantages in the detection of trace substances in complex matrices
[41,42]. Researchers often design novel molecularly imprinted fluorescent sensors according to the characteristics of the target and the environment. By preparing sensors with different numbers of fluorophores and different signal expression modes to detect target analytes, the interference of environmental factors can be greatly reduced, so as to obtain better detection limit and linear range
[43,44]. By selecting appropriate fluorophores and corresponding detection substances, researchers can achieve the goal of detecting two or more different target substances at the same time, which greatly saves the detection time and cost
[45,46]. According to the number of fluorescence peaks and targets, the development of molecularly imprinted fluorescent sensors can be divided into three stages: the first stage is using a single emission peak to detect a single target; The second stage is the construction of ratiometric fluorescent probes for a single target; And that third stage is a multi-element detection stage base on imprinting fluorescence sen.
3.1 Molecularly imprinted single-emission fluorescence detection
It is relatively simple to construct a molecularly imprinted fluorescent sensor based on a single emission peak to detect a single target, which has been widely used in the detection of pesticide residues, metal ions, environmental endocrine disruptors and environmental pollutants
[47]. At present, there are two common preparation methods: one is to use fluorescent functional monomers instead of conventional functional monomers for imprinting. The fluorescent functional monomer has a recognition unit for a target molecule and simultaneously has a fluorescent signal group. The method involves a complex organic functional monomer synthesis process, and the organic fluorescent functional monomer has photobleaching property, so the application is relatively less. The other is to use fluorescent nanoparticles, such as CDs and QDs, as fluorescent signal sources, and to construct imprinted fluorescent sensors by imprinting or doping on the surface of fluorescent nanoparticles. Because of the small size of fluorescent nanoparticles, the molecular imprinting layer is directly coated on the surface of fluorescent nanoparticles, which is not easy to control, so the application is less, and most of them are directly doped with nanoparticles.
3.1.1 Fluorescent functional haplotype
From the point of view of molecular imprinting and fluorescence quenching mechanism, the direct use of fluorescent functional monomers will ensure that the target substance in each imprinting cavity can be better combined with fluorescent functional monomers, thus further improving the sensitivity of fluorescent sensors. Wagner et al. Designed and synthesized a polymerizable fluorescent functional monomer based on naphthalimide, which contains urea binding sites and can respond to carboxylate-containing guests
[48]. By apply that fluorescent functional monomer, the high-selectivity fluorescence detection of the benzoate is realize. Haupt et al. developed an evanescent wave optical fiber sensor by coating a molecularly imprinted polymer containing a fluorescent signal group on a 4 cm-long polystyrene optical waveguide, which was also based on a fluorescent monomer of naphthalimide and a molecule containing a carboxyl group
[49]. The sensor can selectively detect 2,4-dichlorophenoxyacetic acid and citric acid with detection limits of 10
-9mol/L and 10
-6mol/L, respectively. Syu et al. Also prepared a molecularly imprinted fluorescent sensor based on naphthalimide fluorescent functional monomers and successfully identified creatinine molecules
[50]. In addition to using naphthalimide as a fluorescent functional monomer, many domestic research groups have designed and synthesized fluorescent functional monomers based on fluorescein and dansyl methacrylate. Yan et al. Used allyl fluorescein as a fluorophore and introduced the fluorophore into the polymer matrix by precipitation polymerization to design a fluorescent functional monomer, which realized the highly sensitive detection of cyhalothrin
[51]. Wu et al. Synthesized the fluorescent functional monomer of dansyl methacrylate, and prepared the molecularly imprinted fluorescent sensor for specific recognition of bisphenol A by combining its phenolic group with bisphenol A
[52].
Compared with the molecular imprinting sensor prepared by the common functional monomer, the molecular imprinting sensor prepared by the fluorescent functional monomer is characterized in that a fluorescent substance is directly fixed on a recognition site through a chemical bond, and the conversion of a fluorescent signal is more efficient, so that the sensitivity is greatly improved. However, this method requires the complex synthesis of fluorescent functional monomers, which has the disadvantages of narrow excitation peak, broad tail emission peak, poor stability and easy photobleaching. Organic fluorescent functional monomers have been gradually replaced by other new fluorescent materials, so they are rarely used.
3.1.2 Fluorescent nano-particle doped type
QDs, CDs, noble metal nanoclusters, etc. Have good photostability, bright photoluminescence, high quantum yield, narrow emission, broad excitation, long fluorescence lifetime, large extinction coefficient and large Stokes shift, and are often used as fluorescent nanoparticles for the preparation of molecularly imprinted fluorescent sensors
[53].
Researchers can prepare single-emission molecularly imprinted fluorescent sensors by directly imprinting on the surface of fluorescent nanoparticles through surface imprinting technology. Bhogal et al. Synthesized a surface molecularly imprinted polymer layer with high selectivity for ketoprofen on the surface of silane-functionalized cadmium sulfide CDs by sol-gel method. Through the selective quenching of the fluorescence signal of the carbon dot, the surface molecularly imprinted polymer layer had a linear response to ketoprofen in the concentration range of 0. 039 ~ 3. 9 μmol/L, with a detection limit of 0. 01 μmol/L
[54]. Li et al. designed and synthesized a surface molecularly imprinted fluorescent probe on the NaYF
4:Yb,Er of upconversion nanomaterials, using perfluorooctane sulfonate as a template molecule and N, O-bis (trimethylsilyl) trifluoroacetamide as a functional monomer. Through the quenching of the fluorescence of the upconversion nanomaterials, perfluorooctane sulfonate can be efficiently identified and highly sensitively detected in the concentration range of 0.01-15 nmol/L, with a detection limit of 0.01 nmol/L
[55]. The fluorescent nanoparticles are generally only about several to dozens of nanometers, the smaller size makes it difficult to control the imprinting layer coated on the surface of a single fluorescent nanoparticle, and the prepared imprinted fluorescent nanoparticles have smaller particle size, are difficult to separate, and are difficult to be used repeatedly, so they are used less.
The common method is to directly add the fluorescent nanoparticles into the mixed solution prepared by the molecularly imprinted polymer, and finally obtain the doped imprinted fluorescent sensor in which the fluorescent nanoparticles are doped in the molecularly imprinted polymer. Compared with fluorescent sensing based on fluorescent nanoparticles directly, imprinted fluorescent sensors doped with fluorescent nanoparticles generally have lower sensitivity,The main reasons for this phenomenon are as follows: (1) Spherical particles or irregular particles are the main morphology of imprinted fluorescence sensing, which makes most of the recognition sites located inside the polymer, and the high mass transfer resistance hinders the effective recognition of target molecules. (2) It is difficult to embed fluorescent nanoparticles into MIPs uniformly by direct doping, so it is difficult to ensure that each imprinting cavity can be surrounded by fluorescent nanoparticles, which limits the conversion efficiency from target recognition to fluorescent signals
[56]. Morphology control and adjustment is an important way to improve the sensitivity of single-emission imprinted fluorescence sensing. Therefore, researchers have done a lot of work in morphology control of imprinted fluorescence sensing and improving the conversion efficiency from molecular recognition to fluorescence signal
[57].
Morphology plays an important role in the detection sensitivity of molecularly imprinted fluorescent sensors. In conventional solid spheres (fig. 2A) or irregularly-shaped molecularly imprinted fluorescent sensors, the recognition sites are deeply embedded into the highly cross-linked molecularly imprinted material, resulting in a large mass transfer resistance and an imprinting cavity that is not easily accessible. By improving the molecular imprinting fluorescence sensing morphology, such as mesoporous structure (Fig. 2B), core-shell structure (Fig. 2C), hollow mesoporous structure, etc. (Fig. 2D) and core-shell mesoporous structure (Fig.2 E), the mass transfer resistance can be greatly reduced and the imprinting sites can be more accessible
[58]. Compared with the common spherical particles or irregular particles, the mesoporous and core-shell structures have larger specific surface area and smaller mass transfer resistance, which have a good effect on improving the detection sensitivity of imprinted fluorescence sensing, and have been developed rapidly in recent years
[59][60].
Core-shell molecularly imprinted fluorescent sensors anchor a fluorescent molecularly imprinted polymer layer on the surface of a suitable support material, and then leave a suitable imprinted cavity on or near the surface to provide accessible active sites. Commonly reported supporting carrier materials are silica and ferroferric oxide, among which silica nanoparticles are widely used due to their easily controlled synthesis, high chemical/mechanical stability, biocompatibility, and a large number of hydroxyl groups on their surfaces
[61][62]. Li et al. Assembled silica nanoparticles and carboxyl-terminated cadmium telluride QDs into composite nanoparticles, and then anchored the molecularly imprinted layer on the surface of the composite nanoparticles by sol-gel method and surface imprinting technology.Under the optimal conditions, the fluorescence intensity decreased linearly with the increase of BHb in the range of 0. 02-2. 1 μmol/L, and the detection limit was 9. 4 nmol/L
[63]. Chen et al. First introduced a large number of cadmium telluride QDs to the surface of amino-functionalized silica core through amide bonds, and then used silica nanoparticles embedded with fluorescent cadmium telluride QDs as the core of the supporting material.A molecularly imprinted fluorescent sensor for 2,4-dichlorophenoxyacetic acid was prepared by directly imprinting a silica shell on the surface through a sol-gel method
[64]. Based on the mechanism of fluorescence quenching induced by electron transfer, 2,4-dichlorophenoxyacetic acid can be selectively and sensitively detected with a good linearity in the range of 0. 66-80 μmol/L and a detection limit of 2. 1 nmol/L in 20 min. However, this method is also difficult to ensure the existence of fluorescent nanoparticles near the recognition site. In addition, core supporting materials of other structures, such as Metal-organic frameworks (MOFs), nanowires and nanorods, have also attracted wide attention of researchers
[65][66][67].
Mesoporous molecularly imprinted fluorescent sensors are constructed by mesoporous templates to construct worm-like or hexagonal tunnel channels with internal interconnection, which help target molecules penetrate into imprinting sites. Cetyltrimethylammonium bromide (CTAB) is a common template for the construction of mesoporous structures. Kim et al. introduced fluorophores into the mesoporous structure by encapsulating fluorescent nanoparticles into MIPs, and encapsulated cadmium selenide QDs in the pores of mesoporous silica by using cadmium selenide QDs as a signal sensor.A molecularly imprinted mesoporous fluorescent sensor with bisphenol a (BPA) as the target molecule was prepared, and the sensitivity and selectivity were greatly improved compared with solid spheres or irregular particles
[68]. In addition, the combination of mesoporous structure and core-shell structure has also attracted wide attention of researchers
[69,70].
By combining the core-shell and mesoporous structure with the strategy of the molecularly imprinted fluorescent sensor, the prepared molecularly imprinted sensor with the core-shell and mesoporous structure provides good binding sites for target molecules (ions) and fluorophores used for signal conduction,It is also conducive to the penetration of target molecules into the imprinting sites, so that it has the advantages of large adsorption capacity, high imprinting efficiency and good surface properties, and is widely used in the sensing of environmental pollutants, drugs and active molecules
[71][72][73][74][75].
In order to improve the conversion efficiency from molecular recognition to fluorescence signal, our research group proposed a "functional monomer-free" imprinting method, in which the amino and carboxyl groups on the surface of fluorescent nanoparticles were used as the recognition groups of functional monomers for direct imprinting, and no functional monomers such as methacrylic acid and acrylamide were used in this process. Fluorescent nanoparticles play a dual role in molecular recognition and signal output. For example, amino group-containing APTES are often used as functional monomers in the design of imprinted polymers with high recognition efficiency for trinitrotoluene (TNT). In the design of fluorescent sensors with highly selective detection of TNT, CDs with amino groups on the surface were directly prepolymerized with the template molecule TNT, and then added to the TEOS polycondensation solution to prepare imprinted fluorescent sensors by sol-gel method. According to the method, the recognition site is ensured to be on the surface of the fluorescent nanoparticle, so that efficient output from molecular recognition to fluorescence signal conversion is ensured, and the detection sensitivity is remarkably improved. The functional monomer-free imprinting method reduced the detection limit from 190 nmol/L to 17 nmol/L compared to the control group using the functional monomer APTES
[76]. The functional monomer-free imprinted polymer preparation method is also used for the detection of proteins and other substances
[77].
In addition to changing the morphology of molecularly imprinted fluorescent sensors, the strategy of "signal on" mode is also an effective way to improve the sensitivity. At present, the main mode of molecularly imprinted fluorescent sensors is to achieve signal turn-off through fluorescence quenching. Compared with the signal turn-on mode under low background color and the mode of signal amplification strategy, the fluorescence output signal is smaller
[78]. The detection background can be effectively reduced by switching the signal output mode from "off" to "on", thereby improving the detection sensitivity of the molecularly imprinted fluorescent sensor
[79]. Li et al. prepared a "signal-on" molecularly imprinted fluorescent sensor for the detection of tyrosine phosphopeptide (pTyr peptide)
[80]. Cadmium telluride QDs were detect by imprinting a fluorescent signal on that silica-terminated surface. The fluorescence intensity was enhanced due to the specific rearrangement of the passivated trap States of phenylphosphonic acid (epitope) and pTyr peptide on the surface of cadmium telluride QDs, thus improving the detection sensitivity, with a detection range of 0. 5-35 μmol/L and a detection limit of 0. 37 μmol/L. Guo et al. Prepared a bacterial imprinted polydimethylsiloxane modified film, which can realize the fluorescence detection of the specific opening signal of Staphylococcus aureus
[81]. The fluorescence resonance energy transfer platform of the sensor was developed based on the electrostatic interaction between a citrate-functional copper cluster and a dopamine-stabilized gold nanoparticle. The negatively charged bacteria compete with the copper clusters of citrate and bind to the dopamine-stabilized gold nanoparticles, which leads to the fluorescence recovery of the copper clusters of citrate and realizes the "signal on" mode.
Our group also proposed a signal "turn-on" method, using horseradish peroxidase (HRP) as a model target, based on nanoparticle signal amplification for ultra-sensitive detection of glycoproteins (Fig. 3)
[82]. In the experiment, formylphenylboronic acid was used as the functional monomer, and boronic acid modified magnetic nanoparticles were used as the support matrix for HRP imprinting through the surface-oriented imprinting strategy. Boric acid modified tetrakis (4-carboxyphenyl) porphyrin nanoparticles (BA-TCPP NPs) were captured as signal tags by imprinted magnetic nanoparticles in the presence of HRP based on the borate affinity sandwich method. TCPP NPs are dissolved in thousands of porphyrin molecules, resulting in the amplification of HRP fluorescence signal. In this work, because of the introduction of an amplified "on" fluorescence signal in the protein recognition process, resulting in a low background and a high fluorescence signal value, the linear range of HRP detection is 10
-4~10 mg/L, and the detection limit can reach 0. 042 μg/L, which also has a good recovery and sensitivity in actual urine samples.
In the "on" mode, due to its low optical background and high signal to noise ratio, when the substrate is recognized,The fluorescence is obviously enhanced, because a bright signal appearing in a completely dark background is more easily observed than a signal which is already bright and becomes dark, so that the fluorescence can be obviously observed by the naked eye, and the sensitivity of the detection is improved
[83]. The fluorescence sensor in the "signal on" mode has broad application prospects in practical applications
[84]. The sandwich structure based on "signal on" can significantly improve the detection sensitivity by combining a variety of signal amplification means, but there are also some problems in this method. For example, the doping method completes target detection and fluorescence signal output at the same time of target molecule recognition, and the fluorescence "signal on" detection mode based on the sandwich structure has to go through multiple steps such as adsorption-recognition signal unit-signal unit release, which is more cumbersome than the doping method. In addition, in order to construct the "signal on" detection mode of sandwich structure, it is necessary to modify the recognition unit with specific recognition performance to the target on the signal unit, which can be antibodies and aptamers. The use of highly specific recognition units limits the wide application of this method.
3.2 Molecular imprinting ratiometric fluorescence detection
The single-emission fluorescence sensor has only one fluorescence peak signal change for the target detection substance, and the emission peak is easily affected by various factors unrelated to the analyte, such as background interference of the fluorescence sensor, local concentration, photobleaching, fluctuation of instrument and operation parameters, and the detection accuracy is affected
[85]. Compared with single-phosphor fluorescence sensors, ratiometric fluorescence sensors can eliminate the influence of these extraneous environmental factors due to their high sensitivity and built-in correction function
[86,87].
3.2.1 Binary emission molecular imprint ratiometric fluorescence sensing
Generally speaking, binary emission ratiometric fluorescence sensors can be divided into two categories according to the change directions of the two fluorescence intensities: one is a reference ratiometric fluorescence sensor in which one of the two fluorescence signals remains unchanged without being affected by the analyte, and the other is affected by the analyte; The other is a reversible signal ratiometric fluorescence sensor in which both fluorescence signals are affected by the analyte and have opposite changes.
A reference-type ratiometric fluorescence sensor typically contains two fluorescent substances, a target-sensitive fluorophore and a reference fluorophore. With the change of the target concentration, the fluorescence intensity of the target sensitive fluorophore will change significantly, while the reference fluorophore will not be affected by the analyte and will remain unchanged. Based on the intensity ratio of two fluorescence emission peaks (target sensitive fluorophore/reference fluorophore), the ratiometric fluorescence sensor can achieve accurate quantification of the target
[88]. At that same time, the mix color of the two fluorophores is obviously Chan due to different intensity changes of the two fluorescence signals, and the ratio fluorescence signal output can also realize visual detection
[89].
How to embed the reference fluorophore into the molecularly imprinted polymer to block the interaction with the target analyte is a problem that must be faced in the construction of molecularly imprinted reference fluorescent sensors. Doping the reference fluorophore into the polymer is a relatively direct way. In 2015, our group first proposed to construct a reference ratiometric fluorescence sensor through a core-shell structure
[90]. A reference fluorescent nanoparticle red cadmium telluride QDs is directly embed into a silica nanoparticle core through a reverse microemulsion method, and then that silica embedded with the red QDs is use as a solid matrix,A core-shell mesoporous molecularly imprinted ratiometric fluorescence sensor was constructed with CTAB as the mesoporous template and green cadmium telluride QDs as the sensitive fluorescent fluorophore of the target analyte TNT (Fig. 4A). The fluorescent sensor has excellent selectivity and sensitivity, and in the TNT detection process, TNT can quench the quantum dots on the basis of resonance energy transfer caused by electron transfer,The green cadmium telluride ODs embedded in the mesoporous silica shell were selectively quenched, while the fluorescence intensity of the red cadmium telluride QDs remained constant under the protection of the silica shell, and the color of the solution changed from yellow-green to red-orange. The detection limit of the fluorescent sensor can be as low as 15 nmol/L, showing excellent performance. Wang et al. Designed a binary emission molecularly imprinted core-shell mesoporous microsphere, and green fluorescent cadmium telluride QDs doped into the core of silica mesoporous microspheres were used as reference fluorescent nanoparticles.Other red fluorescent cadmium telluride QDs were embedded around the pores of silica microspheres as analytical labeling targets, which could detect malachite green with high sensitivity, and the experimental results showed that the visible color change from orange to green could be observed with the naked eye
[91].
The molecularly imprinted ratiometric fluorescence sensor constructed by the core-shell method has relatively loose conditions on the selection of the fluorophore, does not need to consider whether the reference fluorophore can respond to the polymer, and only needs to ensure that the reference fluorophore and the target response fluorophor have good resolution on the fluorescence peak, so the method is widely applied. However, the preparation of core-shell structure requires a two-step method of core layer preparation and shell layer imprinting, which is more cumbersome. The reference ratiometric fluorescent sensor can be fabricated by co-doping with appropriate fluorophore in one step.
The co-doping method has simple steps, but puts forward certain requirements on the properties of the fluorophore. Except that the fluorescence emission peaks have good resolution at the same excitation wavelength, only one of the two fluorescence signals can respond to the target, and the other can not respond to the target. For example, Xu et al. used cadmium telluride QDs as the target-sensitive fluorescent nanoparticles, hematoporphyrin as the reference fluorophore, and quantum dots as the response fluorophore to prepare a mesoporous structure reference molecularly imprinted ratiometric fluorescent sensor to achieve high sensitivity and selectivity detection of melamine (Fig. 4B)
[92]. Based on the charge transfer mechanism, the CdTe QDs exhibited fluorescence quenching at 540 nm when melamine was detected, while the hematoporphyrin fluorescence intensity did not change, and the fluorescence color changed from yellow-green to red, resulting in an obvious visual signal output. Under the optimal conditions, the detection limit of the ratiometric fluorescent molecularly imprinted sensor with mesoporous structure was as low as 38 nmol/L, and the recovery rate was 92% -101% in the detection of actual milk powder samples.
Wang et al. prepared a reference-type molecularly imprinted ratiometric fluorescent sensor for detecting BHb by mixing a core-shell molecularly imprinted polymer embedded with and doped with green quantum dots and silica nanospheres doped with red quantum dots in a post-imprinting mixing manner
[93]. The molecularly imprinted fluorescent sensor specifically binds to BHb to quench the fluorescence of green QDs, while the red QDs coated with silica are insensitive to BHb and the fluorescence intensity remains unchanged. The results showed that the continuous fluorescence color change from green to red could be observed by naked eyes, and the detection limit was 9. 6 nmol/L. The method can conveniently control the intensity of the initial two fluorophores in the later stage, but the preparation process is more complicated than the core-shell type preparation. At the same time, the fluorophore used as a reference should ensure that it is not affected by the target and other substances in the detection matrix.
Because one of the fluorescence of the reference signal type fluorescence sensor is not affected by the detected substance, the range of color change is relatively narrow compared with the reversible signal type. The reversible signal ratiometric fluorescence sensor has abundant fluorescence color changes, and has better self-calibration function and detection sensitivity of dual fluorescence detection.
When a ratiometric fluorescence sensor having two reversible signals is used, if the substance to be detected itself has fluorescence, only one fluorescent substance is added, and the reversible signal increases due to the fluorescence of the substance to be detected itself, and the signal decreases due to the added fluorescent substance. If the analyte itself has no fluorescence, it is necessary to add two kinds of fluorescent nanoparticles to achieve a reversible signal change. The addition of the target substance usually results in an increase in the fluorescence intensity of one and a decrease in the other, which can eliminate the external interference unrelated to the analyte, so as to quantitatively detect the target analyte more accurately. Xu et al. used gold nanoparticles as seeds and glucose as CDs precursor to prepare dual-emission nanoparticles with emission peaks at 460 and 580 nm by seed growth co-microwave method, and then used the obtained dual-emission nanoparticles as fluorescent signal source to prepare the ratiometric fluorescent molecularly imprinted sensor specifically recognizing bisphenol A (Fig. 5A)
[94]. Due to the interaction between the target analyte BPA and the fluorophore, the fluorescence of gold nanoparticles was quenched at 580 nm, and the fluorescence intensity of CDs was increased at 460 nm. Based on this change, the fluorescence color changed from orange to green, which was easy to observe with the naked eye, and the detection limit was 29 nmol/L. Higher sensitivity and more pronounced color evolution are exhibited compared to single fluorophore sensors. The feasibility of this method was successfully evaluated by analyzing bisphenol A in water samples, and the recovery rate was 95. 9% ~ 98.9%, and the recovery rate of canned food samples was 92. 6% ~ 98.6%. The preparation of dual-emission fluorescent nanoparticles can effectively shorten the preparation process of the imprinted polymer and optimize the detection conditions. In addition, the close contact of the two fluorophores of the dual-emission fluorescent nanoparticles makes the resonance energy transfer between the two fluorophores easier to achieve.
For the detection of the target analyte bisphenol A, Xu et al. conducted further research, synthesized a molecularly imprinted fluorescent polymer based on the post-imprinting modification of silica with a hierarchical mesoporous structure, and simultaneously anchored CDs and QDs to the molecularly imprinted polymer with a hierarchical mesoporous structure to prepare a ratiometric fluorescent molecular imprinted sensor (Fig. 5B).With the increase of the concentration of BPA, the fluorescence intensity of QDs decreased, while that of CDs increased, and the color of the solution changed from pink to gray and then to blue. The obvious color change could be observed in the whole process, and it was feasible to roughly visually detect the content of BPA
[95]. The prepared ratiometric fluorescent sensor with reversible signal has high sensitivity, the detection limit is as low as 0. 13 nmol/L, and the average recovery is between 96. 4% and 102. 0%.
This way of introducing fluorescent nanoparticles after imprinting may cause QDs to block the channels of template molecules and occupy recognition sites. Moreover, this method has a high requirement for the morphology of the molecularly imprinted polymer, because the fluorescent nanoparticles can only bind to the surface of the spherical or bulk molecularly imprinted polymer, resulting in the recognition sites inside the polymer far away from the fluorescent nanoparticles, and the conversion from molecular recognition to fluorescence signal output will be blocked. But at the same time, the method can reduce the influence of the template on the fluorescence intensity due to being placed in a washing solution for a long time in the elution process, and the fluorescence property is kept good, so that the detection sensitivity is improved, and the method is an ideal strategy for constructing a molecularly imprinted fluorescent sensor with low background, high fluorescence intensity, uniform distribution of fluorescent nanoparticles and high activity of the fluorescent nanoparticles.
3.2.2 Ternary emission molecular imprinting ratiometric fluorescence sensing
Binary emission ratio detection overcomes the disadvantage that single emission is easily affected by environmental factors, which greatly improves the sensitivity. However, most of the fluorescent MIPs sensors with dual emission ratio only provide two kinds of emission.The color synthesis mode mainly includes the mixture of blue and red, the mixture of blue and green, and the mixture of green and red, etc. Through the recognition of analytes, they show changes in their respective transition colors, namely blue-purple-red, blue-blue-green, and green-yellow-red. Their visualization function is much higher than the "on" or "off" fluorescence brightness change of single-emission molecularly imprinted fluorescence sensors. In order to further expand the range of color variation and show more abundant fluorescence colors, researchers have proposed ternary emission ratio fluorescence detection. A three-color emissive sensing system is constructed by mixing ternary emissive MIPs in an optimized ratio. This multi-modal sensing platform has better self-calibration function for multiple fluorescence detection.
Yang et al. propose a three-color emission (blue, green, and red emission) fluorescent MIPs sensor (fig. 6) for selective detection of BHb
[96]. The combination of blue, green, and red emissions is optimal for the construction of a triple emission sensor, and all colors can be mixed from the three primary colors of color light. Blue 7-hydroxycoumarin, green cadmium telluride QDs, and red cadmium telluride/zinc sulfide QDs were embedded in BHb imprinted polymers by sol-gel method, respectively, and used as response signals to provide blue, green, and red fluorescence, respectively. A three-color emissive sensing system was constructed by simply mixing the blue, green, and red emissive MIPs in an optimal ratio. After BHb recognition, the blue fluorescence was enhanced, while the green and red fluorescence were quenched, resulting in a gradual increase in the proportional fluorescence intensity of I<sub> blue </sub>/I<sub> green </sub>/I<sub> red </sub > with increasing BHb. The change of the intensity ratio can directly express the fluorescent color evolution, so that the BHb can be visually detected by naked eyes, and the visualization and the operation efficiency are remarkably improved.
When constructing a ternary emission ratiometric fluorescence sensor, the introduction of a reference signal fluorophore that is not affected by the analyte is often not conducive to expanding the color evolution. If a certain color does not change, the evolution of the color will always be affected by this color, and the range will be narrowed. In general, three fluorophores are quenched at the same time, or two are quenched and the other is unchanged, but these go against the attempt to achieve a rich color change. Therefore, it is very important to find three kinds of fluorescent substances that will respond to the target analyte. Yang et al. proposed a triple-emission MIPs sensor for the detection of folic acid by a post-blotting mixing method
[97]. The green fluorescent MIPs and the red fluorescent MIPs are respectively pre-synthesized and then mixed according to a proper volume ratio. The green and red emissions of the two QDs were quenched at different rates, and the blue fluorescence of folic acid itself was gradually enhanced. The asynchronous changes of these three colors make the overall fluorescence color change significantly in the yellow-orange-red-purple-blue range, which provides an excellent tool for ordinary people to accurately detect folic acid visually. Although ternary emission ratiometric fluorescence sensing can provide a wider range of color changes, the preparation and system are more complex, and the ratio of the intensity changes of the three fluorescence signals with the target needs to be optimized repeatedly, which increases the workload of optimizing experimental conditions.
3.3 Molecularly imprinted multicomponent fluorescence detection
Single-target molecularly imprinted sensors are the focus of scientific research because of their relative simplicity and ease of preparation. However, when the sample is complex and contains a variety of targets to be detected, the single detection cycle will be longer, so the development of multiple detection is of great significance
[98]. Luo et al. mixed two independently prepared molecularly imprinted fluorescent sensing microspheres to realize the simultaneous detection of two viruses in human serum. The detection limits of the sensor for hepatitis a virus and hepatitis B virus were 3. 4 and 5. 3 pmol/L, respectively, with the same excellent recovery rate as in a single mode, showing the potential of simultaneous detection of multiple viruses in practical applications
[99]. Wei et al. Prepared molecularly imprinted polymer shells of norepinephrine and epinephrine on the surface of two different color quantum dots without spectral overlap, and then mixed them to achieve the purpose of simultaneous detection of the two substances
[100]. The experimental results show that the detection limits of the sensor for norepinephrine and epinephrine are 9 nmol/L and 12 nmol/L, respectively, showing excellent performance. However, the mixed binary detection method does not reduce the preparation process of the imprinted polymer, and can not be called a real binary detection fluorescence sensor. In recent years, Chen Lingxin of the Chinese Academy of Sciences has achieved simultaneous detection of multiple target substances in complex water samples based on microfluidic paper chips. For example, they proposed a novel three-dimensional origami ion imprinted polymer microfluidic paper chip device, which transfers the liquid phase ion imprinted polymer to solid glass fiber paper and improves the portability of the device
[101]. Simultaneous detection of both Cu
2+ and Hg
2+ ions was achieved in different regions on the solid glass fiber paper with detection limits of 0.035 and 0.056 μg/L, respectively. Similarly, they achieved simultaneous detection of two different phenolic pollutants, 4-nitrophenol (4-NP) and 2,4,6-trinitrophenol (TNP), as well as chromium and lead ions, through a multiplexed rotating microfluidic paper-based chip
[102][103].
3.3.1 Double reference type
A variety of interferents may exist in environmental samples at the same time, and the target molecules (ions) can quench a fluorescent nanoparticle at the same time, while the molecular (ion) imprinted fluorescent sensor can effectively eliminate the interference of other analogues. In order to achieve the purpose of detecting two targets at the same time and make one signal respond to only one target, it is necessary to design two completely independent fluorescence signal response systems to detect two different targets at the same time, so the design of dual-target fluorescence sensors needs to meet strict constraints. First, the fluorescent nanoparticles respond to only one target, one target acts on only one fluorescent nanoparticle, and the presence of one target causes one signal to change while the other remains constant as a reference, allowing the two fluorescent nanoparticles to reference each other. Second, the two fluorescent nanoparticles need to have good resolution to ensure that two independent emission peaks appear in the fluorescence spectrum. In addition, fluorescent particles also need to have specific functional groups, which can interact with template molecules to form stable complexes, and the interaction between template molecules and functional groups leads to significant changes in fluorescence intensity.
Our group selected CDs and gold nanoparticles as fluorescence signal sources, and prepared a dual-reference ion imprinting ratiometric fluorescence probe for simultaneous detection of Ag
+ and Pb
2+ (Fig. 7)
[104]. The reason for using CDs and Au NCs is that the maximum emission peaks of CDs and Au NCs are at 430 nm and 600 nm, respectively, and the resolution is good. More importantly, the emission peak of CDs does not coincide with the excitation peak of Au NCs, which are independent of each other. The responsiveness of CDs and Au NCs to different metal ions is checked, the quenching behavior and quenching efficiency of various ions are comprehensively considered, Cu
2+, Hg
2+ and Fe
3+ plasmas which can simultaneously quench CDs and Au NCs fluorescence signal sources or have weak quenching effect are excluded, and Ag
+ and Pb
2+ are adopted as detection targets. Ag
+ can effectively quench CDs, while the fluorescence intensity of Au NCs remains unchanged. At the same time, Pb
2+ can quench Au NCs, while the fluorescence intensity of CDs remains unchanged. The results showed that the Ag
+ was linear in the range of 0. 2-12. 5 μmol/L with a detection limit of 26 nmol/L in the range of 50-900 nmol/L for 86 nmol/L,Pb
2+. The prepared dual-reference ion imprinted ratiometric fluorescence sensor has been successfully applied to the simultaneous detection of Ag
+ and Pb
2+ in river and tap water samples, thus greatly reducing the cost. The recovery rate of actual water samples was 94. 6% -110. 5%. This strategy brings innovation to the signal output mode in the field of imprinted sensors. The dual-reference type binary detection imprinted fluorescent sensor is also used for the simultaneous detection of Fe
3+ and Cu
2+[105].
3.3.2 Dual-channel detection
In order to avoid the overlap of two emission spectra or excitation spectra in the same channel, the two emission peaks of one excitation channel interfere with each other. Using dual-channel dual-emission fluorescent nanoparticles, two targets can also be detected simultaneously from two independent emission channels, and the accuracy is also improved. Lu et al. synthesized blue and red dual-channel dual-emission CDs through a one-step hydrothermal method, and developed a dual-channel mesoporous ion imprinted fluorescence sensor using blue and red CDs as fluorescence signal sources to detect Cr
3+ and Pb
2+ (Fig. 8)
[106]. The emission and excitation peaks of the blue and red CDs are firstly guaranteed to be two completely independent peaks. Secondly, in order to simultaneously detect two metal ions in the dual channel, each target metal ion can only quench one CDs. Fe
3+, Ag
+, and Hg
2+, which can quench both blue and red CDs, are not suitable as target metal ions. The Cr
3+ effectively quenches the blue emitter CDs but not the red emitter CDs. At the same time, the Pb
2+ can quench the red emitter CDs, but can not quench the blue emitter CDs, so the Cr
3+ and Pb
2+ are used as detection objects to verify the feasibility of dual-channel detection. Under the optimal conditions, the calibration curve was linear in the range of 0. 1 ~ 6.0 μmol/L with a detection limit of 27 nmol/L for the blue channel Cr
3+ and in the range of 0. 1 ~ 5.0 μmol/L with a detection limit of 34 nmol/L for the red channel Pb
2+. The prepared dual-channel mesoporous ion fluorescence sensor is applied to the detection of Cr
3+ and Pb
2+ ions in river water and tap water samples, and the accuracy is satisfactory. The measured Cr
3+ and Pb
2+ concentrations were similar to those measured by atomic absorption spectrometry, indicating the feasibility of the dual-channel detection strategy.
Xu et al. Further realized the dual-channel simultaneous analysis of chromium morphology through ion-imprinted fluorescent polymers with core-shell mesoporous structure
[107]. In this strategy, blue CDs were embedded in silica spheres to detect Cr
6+ through the internal filtering effect in the blue channel, and a Cr
3+ imprinted silica layer doped with red CDs was coated on the mesoporous shell to selectively detect Cr
3+ based on electron transfer. When the Cr
6+ in the blue channel was detected, the fluorescence intensity quenching effect at 440 nm varied linearly from 0. 01 to 10. 0 μmol/L, with a detection limit of 3. 8 nmol/L. When the Cr
3+ in the red channel was detected, the quenching effect of fluorescence intensity at 605 nm varied linearly from 0. 1 to 15. 0 μmol/L, and the detection limit was 46 nmol/L. This dual-channel strategy makes it more flexible to detect two targets simultaneously. Due to the rigorous conditions of preparing the multiplex fluorescence sensor, the two fluorescence signal response systems must be independent and not interfere with each other. At present, there are few studies in this area. At present, double-reference and double-channel molecular imprinting fluorescence detection can only detect two substances at the same time, and the simultaneous detection of three or more targets has not been reported, which requires further research by researchers.
4 Application of Molecularly Imprinted Fluorescence Sensor
Molecularly imprinted fluorescent sensors have been widely used in the detection of environmental pollutants due to their high sensitivity, high selectivity, simplicity, rapidity, low cost and rapid response. The application of molecularly imprinted fluorescent sensors in the detection of metal ions, small organic molecules and biomacromolecules in environmental pollutants is shown in Table 2.
4.1 Detection of metal ions
Ion-imprinted polymers (IIPs) are imprinted polymers prepared by using metal ions as templates, which can be used as recognition elements of fluorescent sensors to detect metal ions rapidly and sensitively
[108]. During the synthesis of fluorescent sensors, ions are usually not directly imprinted in the polymer, but form coordination bonds with fluorophores. With the entry of fluorophores into the polymer, the formation and breakage of coordination bonds between fluorophores and ions lead to the change of fluorescence signals, thus achieving the purpose of fluorescent sensing
[123]. At present, most of the ion detection methods are electrochemical methods, and there are few reports on the use of fluorescence detection methods, which are mostly based on the quenching effect of ions on fluorescent nanoparticles.
4.2 Detection of small organic molecules
Compared with ion detection, fluorescence sensing is more used in the detection of small organic molecules. Molecularly imprinted fluorescent sensors have been widely used in the detection of environmental organic pollutants, especially in the detection of antibiotics, pesticide residues and estrogens. Molecularly imprinted fluorescent sensors have a variety of substrates for the detection of small organic molecules, including water samples, animal serum, human serum, urine, saliva, etc., and the detection mechanisms include single emission signal quenching, single emission signal enhancement, reference type, etc., which are flexible and highly sensitive.
4.3 Detection of biological macromolecules
Enzymes or antibodies, as natural recognition substances, can specifically bind to biological macromolecules, but the chemical stability of these substances is low, the preparation process is very complex, and animal experiments are often involved. In contrast, MIPs have more advantages because of their high stability, simple preparation and easy degradation. However, MIPs fluorescent sensors still have some limitations in the imprinting of biomacromolecules, especially the imprinting of biomacromolecules such as proteins, which is a challenging field in the field of molecular imprinting
[124]. (1) Biomacromolecules have large surface area and many reactive groups on the surface, which can cause cross-reaction during molecular recognition. (2) The large volume of biological macromolecules makes it difficult to get in and out of the internal imprinting sites of highly crosslinked polymers, which makes the elution and recognition of macromolecules difficult. (3) Biomacromolecules are insoluble in conventional imprinting solvents, but in water, the hydrogen bonding force between functional monomers and biomacromolecules will be destroyed, which makes the imprinting effect worse. In order to overcome these difficulties, many methods have been developed, such as surface blotting, epitope blotting, imprinting hydrogel and so on, to realize the imprinting of protein molecules.
Surface imprinting makes the molecular recognition sites located at or close to the surface of the polymer, so that the target molecules can freely enter and exit the recognition sites. Therefore, surface imprinting is a good way to solve the imprinting of biological macromolecules. The epitope blotting method does not use biological macromolecules as templates, but uses a small fragment of biological macromolecules, namely epitopes, in the process of imprinting. Compared with the common Western blotting method, the epitope blotting method has the advantages of uniform binding sites, more specific binding sites, less embedded protein molecules and the like. However, because the volume of the original target protein macromolecule is much larger than that of the template molecule, it is still a problem for the protein to enter the recognition site. The imprinted hydrogel method is to prepare the hydrogel with low degree of crosslinking to solve the problem that the large volume of biomacromolecules are difficult to elute due to the small internal pore size of the polymer and the high degree of crosslinking of the molecular imprinting
[125]. Western blotting generally uses surface blotting and dopamine polymerization. Dopamine can adhere to proteins and has biocompatibility. It can self-polymerize to form highly cross-linked polydopamine films in weak alkaline aqueous solution. Dopamine is used as a functional monomer, and the imprinting effect on the protein is good by imprinting on the surface of the polydopamine film.
5 Conclusion and prospect
In this paper, the latest progress of molecularly imprinted fluorescent sensors is reviewed, focusing on the sensors with different numbers of fluorophores constructed according to the characteristics of the target and the environment in which they are located. Single-emission fluorescence sensor is still the commonly used method because of its simple preparation. The built-in calibration function of ratiometric fluorescence sensors can eliminate the influence of environmental factors and provide self-calibration function of fluorescence detection relative to single fluorophore. The binary detection fluorescence sensor can simultaneously detect two or more target substances, and can also save the detection cost and time.
With the development of experimental technology, molecularly imprinted fluorescent sensors have made great progress in sensitivity and selectivity, but their development still faces great challenges and has broad development space: (1) to develop new fluorescent materials that are economical and environmentally friendly, and to prepare green imprinted fluorescent sensors; (2) In the aspect of improving detection sensitivity, the sensitivity of MIPs was improved by adjusting the morphology, imprinting method, detection mechanism and signal transmission method of MIPs, reducing mass transfer resistance, improving recognition efficiency and signal amplification. Compared with the in-depth study of morphology control, the application of signal amplification technology in the field of imprinted fluorescence sensing is still relatively small. In the future work, we can explore the application of various signal amplification methods in imprinted fluorescence sensing. (3) In the aspect of multivariate detection, there are many attempts based on binary detection at present, and there is no report on multivariate detection imprinted fluorescent sensor. In the future, more combinations of detection modes, such as double labeling and double detection modes, can be explored to achieve the purpose of multivariate detection. (4) In the aspect of achievement transformation, the portable device based on molecularly imprinted fluorescent sensor was studied more deeply. At present, most of them are applied in laboratory, and it is necessary to overcome the matrix interference of actual samples when they are applied to the detection of actual samples on a large scale. (5) At present, most of the molecularly imprinted fluorescent sensors are prepared for various organic/inorganic pollutants and biomacromolecules, while cell experiments and in vivo experiments are relatively few. It is necessary to prepare fluorescent sensors with low toxicity and specific recognition of viruses, bacteria and cells, so as to gradually promote the development of imprinting targets from small molecules to cells and living organisms.
National Natural Science Foundation of China(21777065)
Youth Innovation Project for Colleges of Shandong Province(2019KJA021)
Natural Science Foundation of Shandong Province(ZR2020KE002)
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