1 Introduction
With the continuous development of industrialization, the discharge of some illegal wastes has caused heavy metal pollution, and excessive heavy metals have seriously threatened the environment and human health
[1]. Heavy metals can not be biodegraded, they can accumulate in the environment and organisms, and long-term accumulation will cause a series of hazards to the environment and organisms
[2]. When heavy metal ions enter the human body, they can cause protein denaturation and enzyme activity reduction or even complete loss, thus affecting human health and causing some diseases
[3]. Heavy metals such as lead, cadmium, mercury, chromium and arsenic can cause great harm to human body. For example, Cr (VI) is considered to be a human carcinogen with high solubility, mobility and toxicity in the environment
[4]. The maximum allowable emission of Cr (Ⅵ) is limited to 5 μg/L by the World Health Organization
[5]. Therefore, how to detect the content of heavy metals quickly, simply and efficiently is of great significance for life science, environmental chemistry, medicine, agriculture and other related fields.
On the other hand, metal ions play an important role in life. A variety of metalloproteins and metalloenzymes exist in organisms, such as hemoglobin and myoglobin, which are metalloproteins with iron porphyrin as a prosthetic group
[6]; Hemocyanin is a copper-containing metalloprotein
[7]; Zinc finger protein is a DNA-binding protein containing zinc ions
[8]. The content of metal elements is too little or too much, which will have harmful effects on life. For example, iron deficiency can cause iron deficiency anemia
[9]; High levels of aluminum in the human body can cause serious damage to the nervous system and even induce Parkinson's disease
[10]. The content of metal ions has a great impact on life, so it is very necessary to accurately detect the concentration of metal ions in vivo.
For the detection of metal ions in organisms and the environment, a variety of methods have been developed, such as complexometric titration, Atomic Absorption Spectrometry (Atomic Absorption Spectroscopy, AAS), Atomic Emission Spectrometry (Atomic Emission Spectrometry, AES), Atomic Fluorescence Spectrometry (Atomic Fluorescence Spectrometry, AFS), Inductively Coupled Plasma (Inductive Coupled Plasma, ICP) and inductively Coupled Plasma Mass spectrometry (Inductively Coupled Plas ma
[11⇓~13][14][15][16]. These methods have some disadvantages, such as the need for sample pretreatment, the detection is not fast enough and the price is expensive
[17]. Therefore, there is an urgent need to develop rapid, accurate, low-cost and selective metal detection methods
[18].
Peptide-based detection of metal ions is a rapid and simple method developed in recent years. The research on the application of the interaction between peptides and metal ions has attracted much attention
[19]. The polypeptide is a polydentate ligand formed by the dehydration condensation of amino acids. Peptides have the following advantages: (1) There is a mature Synthesis method, which can synthesize peptides with different sequences by Fomc-Solid Phase Peptide Synthesis (SPPS)
[20]. (2) It is a multidentate ligand with multiple coordination atoms, which can quickly bond with metal ions to form very stable chelates
[21]. Therefore, metal ions can be detected with high sensitivity. And (3) different sequences of the polypeptides endow the polypeptides with different structures and properties and different binding capacities with different metal ions, namely different polypeptides have high selectivity for metal ion detection. And (4) the binding capacity of the polypeptide to the target metal ions can be further optimized by changing the amino acid sequence of the polypeptide so as to achieve higher selectivity and sensitivity. (5) good water solubility, is an endogenous bioactive molecule, and has good biocompatibility and cell penetration capability. Therefore, it can be used for metal ion detection and biological imaging in cells and special organelles. Due to the above advantages of peptides, chemical sensors based on peptides have become one of the effective methods for the detection of heavy metal ions. Among them, UV-Vis absorption spectroscopy, fluorescence spectroscopy and electrochemical analysis are widely used and effective detection methods.
2 Polypeptide-based UV-visible colorimetric sensor
The basic principle of quantitative detection by UV-Vis method is Lambert-Beer's law, that is, the absorbance of chromophore in transparent solvent varies linearly with the optical path of sample cell and the concentration of chromophore (A = εbc). There are two kinds of spectrophotometric analysis, one is based on the absorption of UV-visible light by the substance itself; The other is based on the formation of colored compounds. Although some inorganic ions have absorption in the ultraviolet-visible region, their intensity is generally weak, so they are seldom used directly in quantitative analysis. It is one of the most widely used test methods to convert the substance to be measured into a compound with absorption in the ultraviolet-visible region by adding a chromogenic agent for photometric determination. Absorption spectroscopy has significant advantages for the detection of heavy metal ions, such as simple equipment, convenient operation, high efficiency and low cost.
Nanogold refers to tiny gold particles with a diameter of 1 ~ 100 nm, which are dispersed in water in the form of sol, so it is also called colloidal gold
[22]. Gold nanoparticles have excellent optical properties, such as high molar extinction coefficient, especially strong Surface Plasmon Resonance (SPR) properties. In the UV-Vis spectrum, the characteristic plasma absorption peak of gold nanoparticles is between 510 nm and 550 nm, and with the increase of the size of gold nanoparticles or the decrease of the distance between particles, the position of the ultraviolet absorption peak will gradually move to longer wavelengths, that is, red shift occurs
[23]. When observed by naked eyes, the color of gold nanoparticles is mainly bright red solution. When the gold nanoparticles are agglomerated to a certain extent, that is, the distance between two particles is less than twice the particle size, the color of gold nanoparticles solution will change significantly, from its original bright red to blue-purple.
In recent years, the UV-Vis colorimetric method of gold nanoparticles based on peptides has attracted much attention. Chen et al. Designed a copper ion colorimetric sensor based on peptide (CGGG) modified gold nanoparticles (p-AuNPs)
[24]. In the presence of Cu
2+, a plurality of p-AuNPs are combined together synergistically to cause the aggregation and precipitation of the p-AuNPs, so that the color of the p-AuN is changed from wine red to colorless (fig. 1), and other metal ions hardly interfere with the detection of copper ions, so that the sensor has good selectivity to the Cu
2+. Li et al. studied a sensitive silver ion detection method, which is based on the interaction of polypeptide (RFPRGGDD) modified gold nanoparticles (AuNPs) with Ag
+ through the amino acid residues on the polypeptide, resulting in the aggregation of AuNPs and color change, so as to determine the Ag
+ through spectral colorimetry
[25]. The method can be used in aqueous solution with a detection limit of 7. 4 nmol/L. Chai et al. Used Cys-GNPs formed by L-Cys and gold nanoparticles to detect Hg
2+[26]. When the Hg
2+ is added into the system, the Cys-GNPs can react with the Hg
2+, so that the Cys-GNPs are aggregated into particles with larger size, and the color of the gold nanoparticle solution is changed. The method has good selectivity for Hg
2+ with a detection limit of 100 nmol/L.
Polypeptide-based gold nanoparticles can also be used for visual detection. For example, Si et al. Modified gold nanoparticles with carboxylated peptide chains (NH
2-Leu-Aib-Tyr-OMe,Aib:α- aminoisobutyric acid) to prepare polypeptide-based functionalized gold nanoparticles sensors
[27]. In the presence of Hg
2+ at room temperature, the sensor can self-assemble into a one-dimensional array of gold nanoparticles without any template. The change of solution color and absorption spectrum caused by self-assembly makes the naked eye detection limit of the sensor for mercury ions in aqueous solution reach the ppm level. Li et al. Synthesized a heptapeptide with the sequence of Cys-Cys-Pro-Gly-Cys-Ala-Arg. The sulfhydryl group of cysteine on the peptide forms an Au-S bond with gold nanoparticles, which reduces the charge on the surface of gold nanoparticles, promotes the aggregation of gold nanoparticles and changes the color of the solution from wine red to blue
[28]. After the addition of the Zn
2+, the polypeptide preferentially binds to the Zn
2+ rather than to the gold nanoparticles, and the color of the solution does not change (fig. 2a). Du et al. Used a similar principle to mix gold nanoparticles with a particle size of 13 nm with peptides, and used colorimetry to detect Hg
2+ sensitively
[29].
Feng et al. In our group designed a heptapeptide-based colorimetric sensor (Cys-Pro-Gly-Cys-Pro-Gly-Cys-NH
2,P7) (Fig. 2b)
[30]. When P7 was added to GNP, the sulfhydryl group of cysteine on the polypeptide chain combined with gold nanoparticles to form Au-S bonds, and gold nanoparticles were connected by different sulfhydryl bonds, which promoted the further aggregation of gold nanoparticles and changed the color of the solution from wine red to blue. However, when Hg
2+ and P7 were added to GNP at the same time, the color of GNP was still red. When other metal ions and P7 are added to GNP at the same time, it will turn blue. Therefore, the biosensor can selectively detect Hg
2+ with a detection limit of 0. 1 μmol/L.
Colorimetric analysis is a very important analytical method in the case of resource shortage, which is easy to observe with the naked eye and does not require complex instrument operation procedures. In colorimetric analysis, that signal in the reaction medium is represen by a visual color change. Due to the unique surface plasmon resonance of gold nanoparticles, gold nanoparticles with different particle sizes show different colors. Based on the binding properties of peptides to gold nanoparticles, the study of spectroscopic colorimetric determination of metal ions by gold nanoparticles based on peptides will receive more attention.
3 Polypeptide-based fluorescent chemical sensor
Common fluorescent chemical sensors are mainly composed of the following parts. (1) The recognition group, also known as the receptor, is the recognition moiety of the species to be tested and can selectively bind to the substrate to be tested; (2) a linker, which is an intermediate connecting a recognition group and a fluorescent group; And (3) a fluorescent group, namely a signal report group, can emit a fluorescent signal which is easy to detect after the recognition group is combined with the analyte, so that the purpose of detecting the target analyte can be achieved by using a fluorescence spectrum.
3.1 The Mechanism of Fluorescent Chemical Sensor
There are three common fluorescence sensing mechanisms of fluorescence sensors.
(1) mechanism of photoinduced electron transfer
Photoinduced Electron Transfer (PET) is one of the common mechanisms in fluorescent chemical sensors
[31]. The sensor based on the PET mechanism is generally composed of an acceptor moiety (bonding group), a fluorescent group and a spacer group, and the acceptor moiety or the fluorescent group is generally used as an electron donor or an electron acceptor. The fluorophore part is the site of absorption and emission of fluorescence, while the acceptor part is used to bind the object. The two parts are separated by the spacer and connected by the spacer to form a molecule, which constitutes a molecular system that can selectively recognize the object and give changes in optical signals. Photoinduced electron transfer reaction refers to the phenomenon of electron transfer between the electron donor and the electron acceptor after the electron donor or the electron acceptor is excited.
When the bonding group of the fluorescent sensor is used as an electron donor and the fluorescent group is used as an electron acceptor, the sensor is excited by illumination,As an electron donor, the bonding group can transfer its electrons in the highest energy level to the empty electron orbit of the fluorescent group in the ground state, so that the electrons excited by light can not return to the original ground state orbit to emit fluorescence, resulting in fluorescence quenching. When the guest is added, the bonding group can be combined with the guest, so that the electron-donating ability of the bonding group is weakened, and the photo-induced electron transfer process is hindered; and at this time, the electrons of the fluorescent group excited by light can jump to the original ground state orbit to emit fluorescence, so that the fluorescence is restored. Briefly, when the sensor is not bound to the analyte, the fluorescence is quenched; When bound to the test substance, the fluorescence is restored. This kind of sensor shows obvious "off" and "on" States of fluorescence, so it is also called fluorescent molecular switch.
(2) Intramolecular (photoinduced) charge transfer mechanism
Intramolecular (photoinduced) Charge Transfer (Internal Charge Transfer, ICT) fluorophores contain both electron donors and electron acceptors for guest recognition
[32]. When the fluorophore is excited by light, the electron donor transfers charge to the acceptor, thereby radiating a fluorescent signal. When the guest is present, the donor or acceptor of the fluorophore binds to the guest, which hinders the intramolecular charge transfer between the donor and acceptor, resulting in a red or blue shift in the fluorescence emission spectrum.
(3) fluorescence resonance energy transfer mechanism
Fluorescence Resonance Energy Transfer (FRET) is different from the other two mechanisms in that it requires a pair of suitable fluorophores to form an Energy donor and an energy acceptor pair.The emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. When they are close to each other to a certain distance in space, the fluorescence energy generated by exciting the donor is just absorbed by the nearby acceptor, so that the fluorescence intensity emitted by the donor is attenuated and the fluorescence intensity of the acceptor fluorophore is enhanced
[33]. The efficiency of energy transfer is related to the degree of overlap between the donor's emission spectrum and the acceptor's absorption spectrum, the relative orientation of the transition dipoles of the donor and acceptor, and the distance between the donor and acceptor. The generation of FRET requires strict conditions to be met: (1) the donor moiety and the acceptor moiety must be sufficiently close (10 100 100 Å); (2) there must be an overlap between the absorption spectrum of the energy acceptor and the emission spectrum of the energy donor; (3) The spatial arrangement between the donor and the recipient must be appropriate.
Peptide-based fluorescent sensors have the advantages of easy synthesis, good water solubility and good biocompatibility, so the application of peptide-based fluorescent sensors in the detection of heavy metal ions has attracted more and more attention. Table 1 lists some of the polypeptide-based fluorescent sensors that have been reported to date.
For polypeptide-based fluorescent sensors, certain amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) can be used as fluorophores due to their intrinsic fluorescence, and can also be modified with exogenous fluorophores such as Dansyl chloride (Dansyl), carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), and acenaphthyl chloride (PySO2).
3.2 Dansyl modified polypeptide
As the most commonly used fluorophore, dansyl chloride can be used to modify polypeptides as a labeling signal, so it has a wide range of applications in the design of fluorescent sensors.
Walkup et al. First designed and synthesized a Zn
2+ fluorescent chemosensor based on zinc finger protein, as shown in fig. 3A
[34]. When the Zn
2+ is added, the Zn
2+ coordinates with two cysteines and two histidines in the polypeptide, which causes the structure of the polypeptide to fold and changes the polar environment of the fluorescent group Dansyl, thus leading to the change of fluorescence emission. The sensor has high affinity to the Zn
2+, and other metal ions do not interfere with the detection of the Zn
2+. However, the application of this sensor is limited by the redox environment due to the easy oxidation of cysteine.
Godwin et al. In 1996 also reported a fluorescence ratio sensor for the detection of Zn
2+[35]. Compare with that sensor reported by Walkup et al., the sensor contain two cysteines and two histidine residues in the polypeptide, but contains two fluorescein (F) group as the donor and lissamine (L) group as the acceptor. In that polypeptide sensor molecule, the distance between the two fluorescent group is relatively far, and the intramolecular energy transfer is weak; When the Zn
2+ is added to the polypeptide sensor, the Zn
2+ is coordinated with two cysteine residues and two histidine residues in the polypeptide, so that the polypeptide chain is folded, and the distance between two fluorescent groups is shortened, so that intramolecular energy transfer can occur between the two fluorescent groups, that is, fluorescence resonance energy transfer can occur in the polypeptide after adding the Zn
2+ (fig. 3B).
Wan et al. Designed a novel peptide fluorescent chemosensor for the detection of Zn
2+ by synthesizing a specific peptide sequence HL(Dansyl-Cys-Pro-Gly-His-NH
2)
[36]. The method has high sensitivity and selectivity to the detection of the Zn
2+ in vitro and in vivo living cells, and the detection limit is about 82 nmol/L. The biosensor has low cytotoxicity and can be used as a chemical sensor for high-selectivity Zn
2+ determination in a biological system.
Wang et al. designed two polypeptide fluorescent sensors based on Dansyl, with the polypeptide sequences of Dansyl-Gly-Trp-COOH (DGT1,1) and Dansyl-Gly-Gly-Trp-COOH (DGTT, 2)
[37]. In the presence of Hg
2+, the fluorescence emission of DGT and DGTT was enhanced and blue-shifted. However, DGT has a significant fluorescence quenching effect on Cu
2+, while DGTT has no fluorescence response to other metal ions. The combination of DGT with Pb
2+ under neutral conditions produces a precipitate. Therefore, this system can be designed as an ion switch by using the effects of DGT on different metal ions.
Donadio et al. Developed a peptide-based fluorescent sensor dH3w(dansyl-His-Pro-His-Gly-His-Trp-NH
2)
[38]. When the Zn
2+ and the Cu
2+ are added, the response of fluorescence enhancement is "on" and the response of fluorescence quenching is "off", respectively, and other common metal ions have little effect on the Zn
2+ and the Cu
2+. The dH3w could penetrate HeLa cells and was used to measure intracellular Zn
2+ and Cu
2+. On this basis, Siepi et al. Further found that the affinity of Hg
2+ for dH3w is much higher than that of Zn
2+ and Cu
2+, about 1000 times that of Zn
2+[39]. When the Hg
2+ and the Cu
2+ are added into the Zn
2+/dH3w system, the strong fluorescence of the Zn
2+/dH3w system is quenched. Therefore, the Zn
2+/dH3w system can be used for the determination of Hg
2+ and Cu
2+ with detection limits of (422 ± 80) and (363 ± 73) nmol/L at pH = 4 and pH = 7, respectively.
In 2015, Wang et al. designed and synthesized a polypeptide fluorescent chemosensor H
2L containing a lysine backbone, in which two amino groups on the lysine molecule are coupled with two cysteines and dansyl groups respectively, and the structure is shown in Figure 4
[40]. The biosensor has high selectivity and sensitivity to Cd
2+ with a detection limit of 52 nmol/L. In the presence of Cd
2+, the fluorescence emission intensity of the H
2L is enhanced due to the photoinduced electron transfer effect. The H
2L has excellent cell permeability and low biological toxicity, and is successfully used for detecting the content of Cd
2+ in HeLa cells. In the following year, Wang et al. Reported a L(Dansyl-Ser-Pro-Gly-His-NH
2) of tetrapeptide fluorescent "switch" chemosensor modified by Dansyl
[41]. When the Cu
2+ was added to L, the fluorescence was quenched, and when the S
2- was added, the fluorescence was enhanced.The sensor had high selectivity and sensitivity to the Cu
2+ and the S
2-, and the detection limits were 88 nmol/L and 75 nmol/L, respectively.
Wang et al. Also reported a polypeptide-based fluorescent chemosensor for the detection of Zn
2+, the polypeptide sequence of which is Dansyl-His-Pro-Gly-His-Trp-Gly-NH
2, and the detection limit for the Zn
2+ is about 97 nmol/L
[42]. Subsequently, Wang et al. Designed a tetrapeptide fluorescent "on" response chemical sensor (Dansyl-Glu-Pro-Gly-His-NH
2) which can sensitively detect Zn
2+[43]. Based on the photoinduced electron transfer mechanism, the sensor can specifically detect Zn
2+ in 100% aqueous solution with a detection limit of 18 nmol/L. It can be used for fluorescence imaging of Zn
2+ in HeLa cells under physiological conditions. Then, they designed a "switch" type polypeptide fluorescent chemical sensor (Dansyl-His-Pro-Gly-Glu-NH
2)
[44]. The addition of Zn
2+ and Cu
2+ to the sensor resulted in the "on" effect of fluorescence enhancement and the "off" effect of fluorescence quenching, respectively. The method is highly sensitive to both Zn
2+ and Cu
2+ with detection limits of 4.9 nmol/L and 15 nmol/L, respectively.
In addition, Wang et al. reported a fluorescent "turn-on" chemical sensor (Dansyl-Glu-Pro-Gly-Cys-NH
2) for the detection of Cd
2+ by tetrapeptidyl
[45]. In HEPES buffer solution, it has a highly sensitive fluorescence recognition for Cd
2+ with a detection limit of 45 nmol/L. Through ion interference experiments, it was found that even in the presence of other competing ions, including Zn
2+, Pb
2+, Hg
2+ and Cu
2+, it had high selectivity for Cd
2+. The sensor has also been successfully applied to bioimaging for the selective detection of Cd
2+ in living cells. In recent years, Wang et al. Have done further research on peptide-based metal ion sensors, and designed and synthesized the following :Dansyl-Ser-Cys-NH
2 of sensors, which have good selectivity for Cd
2+ based on the mechanism of photoinduced electron transfer.The sensor has a detection limit of 23.5 nmol/L (13.8 nmol/L;Dansyl-Ser-Pro-Gly-His-Gly-NH
2) and a detection limit of 17.2 nmol/L (H
2S can be detected by the mixture of the sensor and the Cu
2+).Dansyl-Ser-Lys-Ser-Dansyl, two Ser-Dansyl fluorophores were linked by Lys as an intermediate linker, and the binding with Hg
2+ would lead to fluorescence quenching with a detection limit of 7.59 nmol/L;Dansyl-Gly-Cys-NH
2. As a "switch" fluorescence sensor, the addition of Cd
2+ and Cu
2+ showed the "on" and "off" of fluorescence, respectively, with detection limits of 14.5 nmol/L and 26.3 nmol/L, respectively
[46][47][48][49].
Joshi et al. synthesized three heptapeptide fluorescent sensors :Dansyl-Cys-Gly-Gly-His-Gly-Gly-Glu-NH
2(GG2),Dansyl-Cys-Pro-Gly-His-Pro-Gly-Glu-NH
2(PG2) and Dansyl-Cys-Gly-Gly-His-Pro-Gly-Glu-NH
2(PG1) with the general formula of Dansyl-Cys-X-Gly-His-X-Gly-Glu-NH
2)(X=Pro,Gly)
[50]. It was found that the secondary structure of the polypeptide had an important effect on the selectivity of metal ions. GG2, which contained two Gly-Gly sequences, showed a significant selectivity only for Cu
2+ of common metal ions. In contrast, PG2, which contains two Pro-Gly sequences, is highly selective for Zn
2+. However, PG1 containing a Pro-Gly sequence showed selective fluorescence "off" and "on" detection for Cu
2+ and Zn
2+, respectively. On this basis, PG1 was modified onto PEG-PS resin to achieve the purpose of selective detection of Cu
2+ and Zn
2+[51]. In addition, the resin can be effectively reused by simply washing and filtering the resin with EDTA.
Kim et al. Designed a disulfide-linked symmetrical peptide fluorescent chemosensor DPG1 containing a double histidine residue for the detection of Ag
+[52]. The fluorescence intensity of DPG1 was significantly increased by Ag
+, while the fluorescence intensity of DPG1 was quenched by Cu
2+ and Hg
2+. Because the affinity of DPG1 for Ag
+ is much greater than that for Cu
2+ and Hg
2+,DPG1 can detect Ag
+ sensitively even in the presence of Cu
2+ or Hg
2+. It has also been used for the determination of Ag
+ in silver nanoparticles and living cells.
In et al. Synthesized a polypeptide fluorescent chemosensor for the detection of Al
3+, the peptide sequence of which is Dansyl-Ser-Glu-Glu-NH
2[53]. The peptide biosensor is highly sensitive to the Al
3+, and the fluorescence intensity of the peptide biosensor is enhanced due to the coordination of the free carboxyl group of Glu residue, the free hydroxyl group of Ser residue and the oxygen atom of dansyl group in the peptide sequence with the Al
3+, and the detection limit of the polypeptide biosensor is 230 nmol/L. Jung et al. designed a Cu
+ specific fluorescent chemosensor with a polypeptide sequence of Dansyl-Lys-Lys-Cys-NH
2, which binds to Cu
+ mainly through sulfhydryl, sulfanilamide and amide groups on cysteine
[54]. It can be used to detect Cu
+ in aqueous solution, living cells and Golgi apparatus sensitively.
Azuma and Fukushima have designed two kinds of Cys-containing dipeptide fluorescent sensors (DNS-γGlu-Cys, DNS-Cys-Glu)
[55]. The results showed that Hg
2+ had a strong fluorescence quenching effect on DNS-Cys-Glu, but had no effect on DNS-γGlu-Cys. This is because the adjacent sulfhydryl and sulfimide groups in DNS-Cys-Glu can combine with Hg
2+, resulting in electron transfer and fluorescence quenching. Based on this, DNS-Cys-Glu showed the ability to detect Hg
2+ with high sensitivity.
Zhang et al. Synthesized a polypeptide-based fluorescent chemosensor Dansyl-Ser-Pro-Gly-His-Trp-Gly-NH
2(DSH)
[56]. DSH is based on the fluorescence resonance energy transfer (FRET) from Trp (donor) to Dansyl (acceptor) to detect Zn
2+,DSH, and shows strong fluorescence response to Zn
2+ both in vitro and in vivo. In vitro, DSH was able to distinguish zinc from other metal ions with a detection limit of 124 nmol/L. In vivo, DSH can penetrate HeLa cells and enhance the fluorescence response by binding to Zn
2+, indicating that the sensor can be used to detect Zn
2+ in complex biological systems with low cytotoxicity.
Li et al. In our group synthesized a peptide fluorescent sensor Dansyl-Cys-Pro-Gly-Cys-Trp-NH
2(D-P5) for detecting Cd
2+ by Fmoc solid phase synthesis
[57]. D-P5 has high selectivity for Cd
2+, and the fluorescence emission intensity of D-P5 can be significantly enhanced by resonance energy transfer (FRET) and chelate enhanced fluorescence (CHEF) in the presence of Cd
2+. Similarly, Wang et al synthesized a Dansyl-Cys-Pro-Pro-Cys-Trp-NH
2, which can also specifically recognize Cd
2+ with a detection limit of 11.5 nmol/L
[58]. Pang et al. designed a bifunctional peptide fluorescent ratiometric sensor (Dansyl-His-Pro-Gly-Trp-NH
2,D-P4) capable of detecting both Cu
2+ and Hg
2+[59]. The binding of D-P4 to Cu
2+ quenched its fluorescence (fig. 5), whereas the binding of D-P4 to Hg
2+ produced a fluorescence ratio-enhanced response, with no interference from other metal ions. The detection limits of D-P4 for Hg
2+ and Cu
2+ are 37 and 105 nmol/L, respectively.
Then, we synthesized a polypeptide-based multifunctional fluorescent sensor, Dansyl-Gly-His-Gly-Gly-Trp-COOH (D-P5)
[60]. The binding of D-P5 with Hg
2+ results in the folding of D-P5 conformation, which shortens the distance between tryptophan and Dansyl, and the fluorescence resonance energy transfer occurs between them, resulting in the enhancement of the emission peak intensity of Dansyl. However, the combination of Dansyl with Cu
2+ results in the decrease of fluorescence intensity, which is due to the electron transfer between Dansyl and Cu
2+. The detection limits of the sensor for Hg
2+ and Cu
2+ are 25 and 85 nmol/L, respectively. More interestingly, the selective detection of S
2- was achieved based on the D-P5-Hg and D-P5-Cu systems. The fluorescence intensities of D-P5-Hg and D-P5-Cu systems were quenched rapidly by the addition of S
2-, and the detection limits of S
2- were 0. 22 and 0. 38 μmol/L, respectively. Pang et al. Also designed and synthesized a polypeptide fluorescent sensor Dansyl-Glu-Cys-Glu-Trp-NH
2(D-P4)
[61]. The method has good selectivity for the detection of Hg
2+ with a detection limit of 23 nmol/L. At the same time, the system can be used for the visual detection of biological sulfhydryl groups, and the fluorescence of D-P4-Hg solution becomes more green in the presence of Cys, and the detection limit of Cys is 52 nmol/L.
3.3 Pyrene modified polypeptide
Pyrene, as a very good fluorescent functional group, has dual fluorescence emission (monomer and excimer), and is often used in the design of fluorescent chemical sensors. Thirupathi et al. designed a tetrapeptide fluorescent sensor PySO
2-His-Gly-Gly-Lys(PySO
2)-NH
2 based on perylene fluorophore
[62]. In aqueous solution, it only has a selective fluorescence response to the Hg
2+, and other metal ions do not interfere with the Hg
2+. The Hg
2+ is coordinated with the imidazole group and the sulfonamide group in the sensor, and the interaction of the two pyrene fluorophores in the peptide leads to the fluorescence quenching of pyrene. Jung et al. designed a peptide fluorescent sensor, Pyrene-Cys-Gly-Pro-Cys-COOH (fig. 6), for the detection of Cd
2+ in aqueous solution, urine and living cells, with a detection limit of 23 nmol/L
[63]. Hwang et al. synthesized a dipeptide sensor (Py-Ser-Asp-COOH) based on Pyrene fluorophore to detect Al
3+ in groundwater, tap water and urine through ratio response, with a detection limit of 138.1 nmol/L, which is lower than the concentration of aluminum ions in drinking water stipulated by the United States Environmental Protection Agency (EPA)
[64].
Mehta et al. Designed and synthesized a symmetric dimeric peptide sensor (Py-His-Gly-Gly-Cys-NH
2), in which the histidine His imidazole group plays an important role in the selective and high-affinity binding of Zn
2+[65]. The Zn
2+ in the aqueous solution can be sensitively detected through the ratio fluorescence response, and the Zn
2+ can enter colon cancer cells (RKO) and combine with the Zn
2+ to emit obvious green fluorescence. Mehta et al. Synthesized a polypeptide fluorescent sensor (Py-Trp-Pro-His-NH
2) by simulating the coordination group of Cu
+ in Cu (Ⅰ) -containing metalloproteins
[66]. When a Cu
+ is bound to the two-molecule sensor, the fluorescent sensor exhibits a ratiometric fluorescent response that is sensitive and selective to the Cu
+. The binding mode study showed that the imidazole and indole groups on the polypeptide played a key role in the strong binding to the Cu
+. It was successfully used to image the Cu
+ in A549 living cells and organelle Golgi apparatus.
In addition, Jang et al. designed a dipeptide-based pyrene derivative fluorescence chemosensor (PySO
2-Trp-His-NH
2), which has a highly sensitive fluorescence ratio response to Ag
+ and nano-silver ions under physiological pH conditions
[67]. Pyr-WH was able to penetrate living HeLa cells and showed a ratiometric response to intracellular Ag (I).
3.4 FAM/FITC modified polypeptide
Carboxyfluorescein (FAM) and fluorescein isothiocyanate (FITC) are both derivatives of fluorescin, which are often used as fluorescent labels. Compared with FAM, FITC is more active and is the most widely used fluorescein.
Lv et al. designed a novel polypeptide fluorescent chemosensor for the detection of Cu
2+, which was labeled with 6-carboxyfluorescein (6-FAM) fluorophore at the N-terminus and 4- (4 '-dimethylaminophenylazo) benzoic acid (Dabcyl) quencher at the ε-N of Lys at the C-terminus
[68]. In the presence of Cu
2+, the release of the cleavage fragments after the cleavage of the polypeptide chain results in a significant increase in fluorescence with a detection limit of 10 nmol/L for Cu
2+.
Xu et al. Designed a novel peptide fluorescence sensor L(FITC-Ahx-Gly-Cys-Ala-NH
2), which was modified by fluorescein isothiocyanate (FITC)
[69]. The fluorescence of L was quenched by Cu
2+ and Ag
+, while the fluorescence of L was restored by S
2-, completing the "on-off-on" cycle of fluorescence. The detection limits of Cu
2+ and S
2- in L-Cu system are 37 and 15 nmol/L,Ag
+, respectively, and the detection limits of S
2- in L-Ag system are 13 and 70 nmol/L, respectively. The sensor can simultaneously detect Cu
2+, Ag
+ and S
2- in aqueous solution and living cells.
Recently, Wang et al reported a novel peptide-based multifunctional colorimetric and fluorescent sensor (FITC-Ahx-Gly-His-Lys-NH
2,L), which mimics the binding site of Cu
2+ in ceruloplasmin. It has high sensitivity and selectivity to Cu
2+ with a detection limit of 21.6 nmol/L
[70]. Remarkably, the L-Cu system can detect the H
2S by colorimetric and fluorescence changes, and the detection limit is as low as 14.7 nmol/L. In addition, L showed excellent water solubility and low biotoxicity even at high concentrations, and the cell imaging results showed that it could be used to monitor Cu (II) and H
2S in living colon cancer cells (RKO) by confocal fluorescence microscopy imaging.
3.5 Aggregation induce luminescence fluorophore modified polypeptide
The fluorescence of traditional fluorescent chromophores decreases or even does not emit light at high concentrations, which is called "concentration quenching" effect. The main reason of concentration Quenching is related to the formation of aggregates, so the concentration quenching effect is usually called "Aggregation-Caused Quenching" (ACQ). In recent years, Tang Benzhong et al discovered and proposed the "Aggation-Induced Emission" (AIE) effect. They found that some thiophene molecules hardly emit light in solution, but greatly enhance their luminescence in aggregated state or solid film. In recent years, AIE fluorophores such as tetraphenylethylene (TPE) and thiophene have attracted much attention due to their unique luminescent properties.
Using this principle, Neupane et al. synthesized a tetraphenylethylene (TPE) fluorophore-based peptide fluorescence sensor (TPE-Ser-His-CONH
2), as shown in fig. 7
[71]. In the buffer aqueous solution containing NaCl, it has a selective opening response only to the Hg
2+ among 16 metal ions. The fluorescence emission intensity at 470 nm was significantly enhanced due to the aggregation effect after the sensor was coordinated with the Hg
2+. The detection limit of Hg
2+ is 5. 3 nmol/L, which is far below the maximum allowable concentration of Hg
2+ in drinking water stipulated by EPA. On this basis, they further optimized the polypeptide sequence and replaced Ser with Asp. The results show that the selectivity of the sensor for metal ions is related to the nature of the buffer solution
[72]. In distilled water and phosphate buffer at neutral pH, it shows a selective fluorescence aggregation-opening response to Hg
2+, but in neutral Tris and acidic hexamethylenetetramine buffer, it shows a highly selective fluorescence aggregation-opening response to Al
3+. This indicates that the nature of the buffer solution plays an important role in the aggregation-induced luminescence effect of the sensor caused by different metal ions.
Liu et al designed and synthesized an aggregation-induced luminescence based peptide sensor (TPE-Py-EEGTIGYG), which can selectively detect Cu
2+ in aqueous solution and living cells. The polypeptide EEGTIGYG has the dual functions of improving water solubility and specifically binding to cell membranes
[73]. The self-assembly of TPE-Py-EEGTIGYG in high concentration (25 μmol/L) aqueous solution resulted in the formation of nanoaggregates, which exhibited strong fluorescence due to the restricted intramolecular rotation of the benzene ring on TPE-Py. The fluorescence can be obviously quenched by Cu
2+, but not by other metal ions, so that the selective detection of the Cu
2+ in the water is realized. TPE-Py-EEGTIGYG exists as a single molecule in the aqueous solution of low concentration (5 μmol/L) of TPE-Py-EEGTIGYG and its fluorescence is very weak. However, when it binds to the cell membrane, its fluorescence intensity is greatly enhanced, and this fluorescence can be selectively quenched by Cu
2+. Therefore, it can be used to measure the concentration of Cu
2+ in and out of cells.
In addition to TPE as an aggregation-inducing fluorophore, Neupane et al. Synthesized a new aggregation-inducing fluorescent molecule
[74]. A sensor was designed and synthesized based on the fluorescent molecule, as shown in fig. 8. In Tris buffer solution, the Asp group in the molecule specifically binds to the Al
3+, causing the aggregation-induced fluorescence effect, resulting in an increase in the red fluorescence emission intensity at 600 nm, accompanied by a decrease in the fluorescence emission intensity at 535 nm. The Al
3+ can be sensitively detected by using the ratio fluorescence response property.
3.6 Fluorescent sensors for other polypeptide
In addition to the above types of peptide-based fluorescent sensors, nano-ion modified peptide and cyclic peptide fluorescent sensors have also been reported in recent years. Lin et al. reported a novel peptide functionalized carbon dot sensor, f-CDs, for the detection of Ca
2+[75]. The fluorescence of f-CDs was quenched after binding to Ca
2+, and the quenching intensity was linear with the concentration of Ca
2+ in a wide range. The biosensor has good biocompatibility and little cytotoxicity.
Viswanathan et al designed and synthesized a tripeptide (Gly-Gly-His) linked fluorescent hybrid nanoparticle sensor
[76]. In the presence of Cu
2+, the histidine imidazole ring on the tripeptide in the fluorescent hybrid nanoparticle sensor can form a six-membered ring chelate with the Cu
2+, which triggers energy transfer and leads to fluorescence quenching. The detection limit of the sensor for Cu
2+ was as low as 0. 5 μmol/L.
Yang et al. designed and synthesized a novel cyclic peptide fluorescent sensor for detecting UO
22+. The structure of the cyclic decapeptide is shown in fig. 9
[77]. They first studied the fluorescence response of cyclopeptide A to UO
22+, and the results showed that the fluorescence of tryptophan at 360 nm was quenched by UO
22+, but other metal ions such as Th
4+, Nd
3+ and Cu
2+ were also found to cause fluorescence quenching. Therefore, cyclopeptide B was further designed, i.e., the histidine in cyclopeptide A was replaced by phosphorylated serine to obtain the binding ability of the UO
22+ to the phosphate group; Aspartic acid is replaced by glutamic acid, because the weak acidity and flexible side chain of glutamic acid will enhance the binding ability with UO
22+. The results showed that the fluorescence of cyclopeptide B was quenched by the binding of UO
22+ to cyclopeptide B, while other metal ions did not interfere. Cyclic peptide B has high selectivity and sensitivity for the detection of UO
22+, and has been successfully used as a fluorescent sensor for the detection of UO
22+ in river water.
4 Polypeptide-based electrochemical sensor
Electrochemical analysis is a rapidly developing method in recent years, which is based on classical polarography, and on this basis, oscillographic polarography and anodic stripping voltammetry are derived
[78]. It is an analytical method based on the electrical and electrochemical properties and changes of the solution of the substance to be measured. This kind of method usually uses the test solution as the electrolyte solution to select appropriate electrodes to form an electrochemical cell, and the analysis results can be obtained by measuring some electrical parameters of the electrochemical cell, such as conductance, potential, charge and current, or by measuring the changes of these electrochemical parameters in a certain process. In recent years, great progress has been made in peptide-based electrochemical methods for the detection of metal ions.
Gooding et al. Used reduced glutathione (GSH) modified electrode to detect Cd
2+ and obtained the detection limit of 5 nmol/L
[79]. GSH has no electrochemical activity, which limits its application in the electrochemical detection of heavy metal ions in solution. Liu Younian et al. selected ferrocenyl to modify GSH, synthesized reduced glutathione-ferrocene compound (GSH-Fc) for the first time (Fig. 10), and modified GSH-Fc on gold electrode according to the property that sulfhydryl compound is easy to react with gold to form stable Au-S bond
[80]. Glutathione has many active groups, which can coordinate with heavy metal ions, resulting in the change of electrochemical properties of the electrode. Therefore, the GSH-Fc electrode can be used to detect the Cd
2+, and the detection limit of the sensor is 0.1 nmol/L. Liu Younian et al synthesized an electroactive sensor Fc-GSSG-Fc using oxidized glutathione and ferrocene, and studied its electrochemical properties and coordination with zinc ions by cyclic voltammetry
[81]. As a result, it was found that that bin ratio of the Zn
2+ to the Fc-GSSG-Fc was 1:1.
Chow et al designed four chemically modified gold electrodes, three of which were covalently modified with peptides Gly-Gly-His, γ-Glu-Cys Gly and human angiotensin I through self-assembled lipoic acid monolayers, and the fourth was modified only with lipoic acid
[82]. The electrochemical properties of a mixture of three metal ions, Cu
2+, Cd
2+, and Pb
2+, were measured using these electrodes. The voltammetric study showed that there was an obvious reduction peak, which was attributed to the reduction of copper. There is also a peak attributed to the overlap of cadmium and lead, which is difficult to quantitatively analyze by traditional methods. Therefore, a multi-dimensional partial least squares (N-PLS) regression calibration method was used to analyze the trace concentrations (100 nmol/L ~ 10 mmol/L) of Cu
2+, Cd
2+ and Pb
2+ in the mixture, and satisfactory results were obtained. Lin et al. Reported a peptide-based electrochemical sensor for the selective detection of Pb
2+[83]. Square wave voltammetry (SWV) was used to measure the electrochemical performance of the modified electrode. As shown in fig. 11, the bare gold electrode itself has no obvious electrochemical characteristics. The SWV response was obvious after modification by peptide, and there was a unique peak at 364 mV, which may be caused by the presence of Cys in the peptide. When Pb
2+ was added, a new peak appeared at -307 mV. The sensor has a highly sensitive response to Pb
2+ in the range of 50 ~ 700 nmol/L.
Clara et al. Developed a glutathione modified screen-printed carbon nanofiber electrode (GSH-SPCNFE)
[84]. The GSH-SPCNFE was compared with the classical glutathione modified screen-printed carbon electrode (GSH-SPCE), and the Cd
2+ and Pb
2+ were simultaneously determined by voltammetry. Their electrochemical properties and analytical performance showed that SPCNFE could better support the immobilization of GSH. GSH-SPCNFE has good repeatability and accuracy for the determination of low concentration of Cd
2+ and Pb
2+ in environmental samples.
Liu et al. Developed a simple, sensitive and low-cost electrochemical impedance spectroscopy for the detection of Zn
2+ using unmodified gold nanoparticles (AuNPs) and Zn
2+ specific binding peptides (CCPGC)
[85]. When AuNPs were modified on the glassy carbon electrode (GCE), the peptide containing sulfhydryl group covalently bound to the AuNPs on the electrode, resulting in a large electron transfer resistance. When the Zn
2+ is present, the Zn
2+ and the polypeptide (CCPGC) preferentially bind together due to their high affinity. In this way, that polypeptide can no long interact with the AuNPs on the electrode, and the electron transfer resistance is reduced. Therefore, the Zn
2+ can be quantitatively determined by analyzing the decreased value of the electron transfer resistance.
The sensitivity of this method for the determination of Zn
2+ is comparable to that of atomic absorption spectrometry and higher than that of fluorescence, colorimetric and electrochemical sensors. The method has high ion selectivity, and has great potential in environmental detection and clinical application. Roozbahani et al. developed a fast, real-time and label-free nanopore sensor using aspartic acid-containing polypeptides as chelating agents to control the net charge of polypeptide ligands and their metal ion complexes by adjusting the pH of electrolyte solutions (Fig. 12)
[86]. The concentration of the analyte can be determined by detecting the ionic current generated by the passage of the analyte through a single nanopore. The sensor can detect Th
4+ with a detection limit of 0. 45 nmol/L. The sensor has high metal ion selectivity,Ven if the UO
22+ is 10
3 orders of magnitude higher than the concentration of the Th
4+,Pb
2+ 、Cu
2+ 、Ni
2+ 、Hg
2+ 、Zn
2+ 、As
3+ 、Mg
2+ 、And that Ca
2+ doe not interfere with the detection of the UTh
4+. The method has been successfully applied to the analysis of simulated water samples.
5 Conclusions and outlook
Metal pollution in the environment, especially toxic heavy metals, is a serious threat to the ecological environment and human health. On the other hand, trace metals play important biological functions in organisms, and the imbalance of trace metals leads to the occurrence of many diseases. It is very important for the accurate determination of environmental and biological metal ions. Therefore, the design and synthesis of novel metal ion sensors with high sensitivity, high selectivity and good biocompatibility are still one of the hot topics in chemistry and biomedicine. In recent years, the rapid development of peptide-based metal sensors is due to the following advantages: the synthesis method of peptides is relatively mature, and peptides with different sequences can be synthesized by Fomc-solid phase peptide synthesis method; The polypeptide can be rapidly bonded with the metal ions to form a very stable chelate so as to ensure high sensitivity to the detection of the metal ions; Peptides with different sequences have different binding abilities to different metal ions, which determines the high selectivity of metal ion detection. The amino acid sequence of the polypeptide can be optimized to improve its selectivity and sensitivity; The polypeptide has good biocompatibility and cell penetration ability. Therefore, peptide-based chemical sensors have become one of the effective methods for detecting heavy metal ions in the environment and metal ions in living cells and metal biological imaging. In this paper, we reviewed some peptide-based metal ion sensors reported in recent years, including peptide-based UV-Vis absorption colorimetric sensors, peptide-based fluorescence sensors and peptide-based electrochemical sensors.In particular, the detection of heavy metal ions in environmental pollution (such as Hg2+, Cd2+) and bioessential metal ions (such as Cu2+, Zn2+) and their applications in biological imaging were summarized. Although polypeptide-based sensors have made some important progress in metal ion detection and biological imaging, there are still some problems to be further studied and explored. For example, the specificity of polypeptide-based metal ion sensors still depends on the difference in the binding capacity of the polypeptide to different metal ions, which means that some interference from other metal ions is inevitable; The design of peptide-based metal ion sensors is still at an immature and empirical stage. At present, the main research is the peptide-based fluorescent sensors, and the research of other types of peptide-based sensors is less. The reported peptide-based sensors still fall short of commercial requirements.
In view of the current research situation, we can start from the following aspects to further study and develop new peptide-based metal ion sensors and explore their applications. (1) design and synthesizing a high-selectivity and high-sensitivity sensor by simulate that active binding sites of metal ions in natural metalloprotein/metalloenzyme to realize the specific bin of the polypeptide and the metal ions. (2) Use computer-aided design or theoretical calculations to design more effective specific sensors. (3) In addition to the common fluorescent sensors, other types of peptide-based sensors need to be further strengthened. Innovate the design principle of sensor and develop new detection mechanism. (4) In addition to fluorophores, polypeptide-based metal sensors using other materials such as nanoparticles, polymers, quantum dots, carbon dots, graphene and near-infrared dyes as signal labels were further studied. (5) To study polypeptide-based metal sensors with biological targets such as tumors, cells and organelles. (6) To develop a polypeptide sensor that is portable and meets the commercial needs and the function of multi-metal detection based on chip technology. It is believed that with the continuous exploration and exploration of peptide-based metal sensors, new methods and strategies will be found to further promote the development and application of metal sensors.
the National Natural Science Foundation of China(20471025)
the National Natural Science Foundation of China(21142003)
the National Natural Science Foundation of China(21977042)
the Scientific Research Foundation of Liaocheng University(318011919)
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