1 Introduction
DNA biosensor has high specificity, which has been the research focus in the field of biosensing
[1⇓~3]. Traditional DNA biosensors have some limitations in the immobilization of DNA probes and the output of sensing signals, which reduce the sensitivity of detection
[4⇓~6]. In the past 20 years, DNA biosensors based on nanomaterials, such as metal or metal oxide nanoparticles, quantum dots, carbon nanoparticles, inorganic-organic composite nanomaterials and rigid DNA nanomaterials, have gradually improved their detection sensitivity due to the rapid development of nanomaterial science and its wide application in the field of biosensing
[7⇓⇓~10][11⇓⇓⇓⇓~16][17⇓~19][20⇓~22][23⇓~25][26⇓~28]. Compared with traditional DNA biosensors, DNA biosensors based on nanomaterials can not only increase the effective immobilization rate of DNA probes, but also improve the output rate of DNA hybridization signals, which greatly improves the sensitivity of detection
[29⇓~31]. Among the nanomaterials used in DNA biosensors at the present stage, mesoporous silica has been widely used to amplify the detection signal to improve the sensitivity of DNA biosensors because of its adjustable pore size, large pore volume, large specific surface area, easy surface modification, stable chemical properties and good biocompatibility.
In 1992, scientists from Mobil Corporation first reported MCM-41 ordered mesoporous silica nanoparticles (MCM-41 MSNs) with uniform pores, high specific surface area and controllable pore size
[32,33]. MCM-41 MSNs are usually synthesized from an alkaline precursor solution containing a silicon source, a template agent, a catalyst, a solvent and the like by related technologies (such as hydrothermal treatment and microwave heating treatment) under proper parameter adjustment
[34,35][36,37]. The synthesized MCM-41 MSNs not only have the hexagonal structure of one-dimensional uniform ordered pore structure, spatial symmetry and unidirectional void system, but also have the surface that can be easily functionalized and modified and the pore size that can be regulated
[38,39][40][41,42]. In addition, MCM-41 MSNs showed good hydrothermal stability and biocompatibility
[43]. By combining the prepared MCM-41 MSNs with DNA molecules, signal probes, active nano-materials and electrodes, a variety of multifunctional MCM-41 mesoporous silica nanocomposites for efficient sensing detection can be constructed, and high-sensitivity detection of target substances can be realized. In order to better combine MCM-41 MSNs with DNA biosensors, scientists have explored various forms of MCM-41 MSNs (such as spherical and film-shaped) and their corresponding ordered mesoporous structure functions (such as the controlled release function of orifices)
[44][45][46].
In recent years, MCM-41 MSNs are mainly used in various fields in the form of carrier functions
[47,48]. In the field of DNA biosensing, we mainly focus on three functions of MCM-41 MSNs to expand the sensitivity of detection, namely, as the delivery system of signal probes, molecular sieves and carriers of active nanomaterials
[49,50][51][52].
In this paper, the synthesis methods of MCM-41 MSNs (spherical and thin film) in recent years and the common methods of template removal were summarized, and the methods of surface functionalization modification were introduced. Then, the DNA biosensors using MCM-41 MSNs as the delivery system of signal probes, molecular sieves and active nanomaterials as the carriers to improve the detection sensitivity are systematically described. Finally, the development prospect of MCM-41 MSNs in the field of DNA biosensing was prospected.
2 Synthesis of MCM-41 Mesoporous Silica Nanoparticl
The synthesis of MCM-41 MSNs is generally based on the supramolecular self-assembly of silica precursors (soft template method). The process can be divided into five steps: (1) adding surfactant into alkaline medium solution to form round micelles
[53]; (2) the round micelles gradually form into cylindrical micelles; (3) subsequently, the cylindrical micelles are packed into a regular micellar liquid crystal array; (4) adding a silicon source into the alkaline medium solution, wherein the silicon source is hydrolyzed into anionic silicate and is adsorbed on the surface of the positively charged micellar liquid crystal array through electrostatic action; And (5) removing the surfactant from the preliminarily prepared MCM-41 MSNs to obtain the MCM-41 MSNs with an ordered mesoporous structure.
MCM-41 MSNs can be obtained in different shapes by using related processing techniques and regulating the parameters of the reaction reagents, among which spherical MCM-41 MSNs and thin film MCM-42 MSNs are the most widely used in the field of DNA biosensing
[54][55,56][57]. The synthesis strategies of these two types of MCM-41 MSNs will be briefly outlined below.
2.1 Spherical MCM-41 mesoporous silica nanoparticles
Spherical MCM-41 MSNs are the most typical type of MCM-41 MSNs series, which are usually prepared by sol-gel method based on St Stöber method
[58]. Based on different synthesis strategies, there are three main preparation methods of spherical MCM-41 MSNs at present, namely hydrothermal method, microwave-assisted synthesis method and room temperature synthesis method
[34,35][36,37][59,60].
2.1.1 Hydrothermal Method
The hydrothermal synthesis of spherical MCM-41 MSNs is to seal the precursor solution containing silicon source and surfactant in an autoclave with a certain high temperature and high pressure, and then use the strong convection in the autoclave to make the precursor solution gradually form a supersaturated solution to crystallize and precipitate spherical MCM-41 MSNs. This method is the traditional method to synthesize spherical MCM-41 MSNs, and the MCM-41 MSNs prepared have considerable monodispersity. However, this method generally requires higher reaction temperature (60 ~ 150 ℃) and longer reaction time (1 ~ 6 days), and the MCM-41 MSNs prepared by this method have the phenomenon of crystal transformation, that is, irregular particles may be obtained.
Xu et al. prepared spherical MCM-41 MSNs doped with vanadium by putting the precursor solution containing silicate, CTAB template and NH
4NO
3 into a Teflon-lined autoclave and heating at 110 ℃ for 48 H
[61]. Zhu et al. sealed the precursor solution containing polyvinylpyrrolidone, catalyst NaOH, template CTAB, and silicon source TEOS in a Teflon-lined autoclave and heated it at 80 ° C. Strong convection in the autoclave was used to crystallize the precursor solution to precipitate MCM-41 MSNs with hollow structure
[62]. Sun et al. sealed the precursor solution containing ketone acetate, NH
3·H
2O, methylcellulose, template agent CTAB and silicon source TEOS in a polytetrafluoroethylene-lined autoclave and heated it at 100 ℃ for 12 H. Under the strong convection of the autoclave, the precursor solution gradually reached supersaturation and finally crystallized out MCM-41 MSNs with hollow structure
[63].
2.1.2 Microwave-assisted synthesis
The synthesis of spherical MCM-41 MSNs by microwave-assisted synthesis is a method that uses the stable energy generated by microwave radiation to uniformly heat the precursor solution containing silicon source and surfactant, and then the product is washed and dried to obtain spherical MCM-41 MSNs. Compared with the traditional hydrothermal method, the method has higher yield, purity and crystallization reaction rate, can obviously reduce the reaction time, and the heating of a reaction system is more uniform. However, this method requires strict experimental equipment, and may have a certain impact on the human body in the process of operation.
Xu et al. put the precursor solution containing silicon source TEOS, template agent CTAB and catalyst NaOH into a microwave oven and heated it for 50 min at a power of 120 W, and then washed and dried the product to obtain spherical MCM-41 MSNs
[64]. Rungsi et al. Obtained spherical MCM-41 MSNs by microwave treatment of precursor solution containing silicon source, template agent CTAB and catalyst NaOH at 70 ℃, and rinsing and drying the product
[65]. Yoon et al. obtained spherical MCM-41 MSNs by putting the precursor solution containing 1,2-bis (trimethoxysilyl) ethane, template agent CTCI and catalyst NaOH into a container lined with polytetrafluoroethylene for microwave heating treatment, and rinsing and drying the resulting particles
[66].
2.1.3 Room temperature synthesis method
The room-temperature synthesis method is a method for preparing spherical MCM-41 MSNs by strictly adjusting parameters and continuously stirring a precursor solution containing a silicon source, a surfactant template and the like at room temperature. The method is simple and does not require expensive equipment, and the spherical MCM-41 MSNs prepared by the method also have good reproducibility. However, this method requires strict control of the ratio of reagent parameters to avoid the generation of spherical particles that are not agglomerated.
Fowler et al. obtained spherical MCM-41 MSNs with hollow structure by stirring the precursor solution of silicon source TEOS, template agent CTAB, ethanol and ammonia water solution with a certain parameter ratio at room temperature, and then filtering, rinsing and drying the generated crystal particles
[67]. Mel Meléndez-Ortiz et al. obtained spherical MCM-41 MSNs by continuously stirring the precursor solution containing silicon source TEOS, template agent CTAB, ethanol and ammonia water with strict parameter ratio at room temperature, and filtering, rinsing and drying the resulting solid particles
[68]. Xie et al. prepared magnetic spherical MCM-41 MSNs with a core-shell structure by dispersing magnetic Fe
3O
4 nanoparticles in a precursor solution containing silicon source TEOS, template CTAB, and catalyst ammonia solution, and then stirring at room temperature for 24 H and washing and vacuum drying the precipitated crystals
[69].
Sol-gel method is the most commonly used method to prepare spherical MCM-41 MSNs, which has the advantages of good control of reaction kinetics, easy composition modification and customizable microstructure. The spherical MCM-41 MSNs prepared by hydrothermal method is the most traditional one among the three sol-gel methods. The spherical MCM-41 MSNs prepared by hydrothermal method also have good monodispersity, but the control of particle crystal type is relatively weak. The microwave-assisted synthesis method is the method with the highest yield and the shortest reaction time, but it is also the method with the highest requirements for experimental operation. The room temperature synthesis method is the method with the lowest experimental cost, but the parameter ratio of reagents in the precursor solution needs to be strictly controlled.
2.2 Film-shaped MCM-41 type mesoporous silicon dioxide nano particle
The synthesis of MCM-41 MSNs in the form of a thin film is generally carried out with the aid of a substrate material. At present, there are four commonly used synthetic methods, namely, the self-assembly technology of Gemini surfactants and organosilicon, the modified St Stöber method, the oil-induced self-assembly technology and the electrochemical assisted method (EASA)
[70][71][72][73]. The basic principles and characteristics of these methods will be briefly introduced in turn.
2.2.1 Self-assembly of Gemini Surfactant and Organosilane
In 2011, Ma et al. synthesized mesoporous silicon films with vertical channels on silicon wafers using NaOH as a catalyst and a pH regulator of the reaction medium, Gemini and CSDAS as surfactants and structure-directing agents, ethanol as a cosurfactant, and TEOS as a silicon source
[70]. Gemini is an amphiphilic molecule with two polar groups, which has high charge density and large group area. Its high hydrophilic/hydrophobic ratio enables it to construct a supramolecular template of mesoporous structure with organic/inorganic interface curvature, so that the prepared film has ordered pores perpendicular to the substrate. The presence of ethanol alleviated the hydrolysis rate of TEOS and the condensation rate of silicate on silicon wafers, resulting in more uniform film growth. However, since the pore walls are composed of polymers and silicones, the thermal stability and mechanical properties of the films are usually low.
2.2.2 Modified St Stöber method
In 2012, Teng et al. prepared a mesoporous silicon film with a vertical uniform pore structure on an ITO wafer by immersing the ITO wafer in a St Stöber solution containing TEOS, CTAB, ethanol and ammonia
[71]. In this method, the ratio of CTAB/TEOS, the ratio of ethanol/water and the concentration of ammonia are the key factors affecting the thickness of the film, and the ratio of ethanol/water can also directly affect the order of the pore structure of the film. However, this method is only suitable for synthesis on ITO wafer at present, and the parameters of experimental reagents need to be strictly controlled.
2.2.3 Oil-induced self-assembly technique
In 2015, Kao et al. Immersed polished silicon wafers or ITO wafers in an oil-in-water emulsion composed of CTAB, ethanol, decane and other oily substances and ammonia water at 50 ℃ to prepare mesoporous silica films with vertically ordered pores
[72]. In this method, decane or other oily substances play a decisive role in the formation of thin film-shaped MCM-41 MSNs, not only as a pore-expanding agent, but also as a structure-directing agent. The concentration of ammonia and ethanol affects the thickness of the film and the order of the mesoporous structure, and this method has no special requirements for the substrate, so it has a certain degree of versatility.
2.2.4 Electrochemical Assisted Approach (EASA)
Film-like MCM-41 MSNs can be synthesized in acidic media by electrochemical techniques (EASA method) in addition to the commonly used alkaline precursor solution. Walcarius et al. First synthesized and reported the preparation of thin film MCM-41 MSNs by electrochemical techniques in an acidic medium
[73]. The EASA method is generally carried out according to the following steps: firstly, an electrode is immersed in a precursor solution containing CTAB, TEOS and the like, and then a certain cathode potential is applied, at this time,Transient electrochemically driven self-assembly occurs on the surface of the electrode and hydroxyl ions are simultaneously generated to catalyze the polycondensation of the silicon source in the direction perpendicular to the substrate electrode so as to form the mesoporous silicon film with a hexagonal mesoporous structure
[74⇓~76]. The method is simple, rapid and economical, and does not require additional chemical modification of the substrate
[77,78]. However, this method is only applicable to a conductive substrate (such as an electrode), and the film produced on a conductive substrate having a large area may not be uniform. In addition, higher electronegativity or longer deposition time can lead to the formation of additional silica aggregates or spheres on the top of the film, thus destroying the uniformity of the film.
To sum up, the self-assembly of Gemini surfactant and organosilane is the simplest of the four synthesis methods, which can effectively prepare mesoporous silicon films with vertical uniform pore structure, but the thermal stability and mechanical properties of the films are relatively low. The modified St Stöber method is the method with the mildest reaction conditions and the relatively best comprehensive performance of the prepared film, so it has quite good application prospects in many aspects, but this method needs to strictly control the parameters of the reagents and longer reaction time. Oil-induced self-assembly is the most versatile method, which can be used to prepare MCM-41 MSNs films with vertical channels on different substrates, and can be widely used in various fields. EASA is the simplest, fastest, and most economical method, and has considerable applications in areas such as electrochemical sensing. However, this method requires strict control of the cathodic potential and electrodeposition time to avoid the formation of additional silica aggregates or spheres on the top of the film. The MCM-41 MSNs thin films synthesized by the four methods all have ordered vertical channels and small pore size (2 ~ 3 nm), so they have been widely used in the field of electrochemical sensing
[79,80].
3 Template Removal of MCM-41 Mesoporous Silica Nanoparticles
The template plays a very important role in the formation of ordered mesoporous structure of MCM-41 MSNs. However, almost all templates will block the mesoporous channels, affecting the order, pore volume and pore size of the mesoporous channels, thereby affecting their capacity as carriers and the controlled release ability of the orifice. Therefore, the prepared MCM-41 MSNs usually require effective template removal. At present, the common methods that can effectively remove the template are high-temperature calcination, solvent extraction, microwave-assisted method, ultrasonic treatment and oxidation
[81][82][83][84][85].
3.1 High-temperature calcination method
Calcination at high temperature is the most traditional method to remove the template. Usually, MCM-41 MSNs containing surfactant molecules are calcined in air or oxygen at 500 ~ 600 ℃ to oxidize the template into CO
2 and H
2O to achieve the effect of template removal
[86⇓⇓~89]. The method does not need complicated operation steps, can save experimental operation time, and can completely remove the template agent in the channel of the MCM-41 MSNs. However, during the calcination process, the continuous high temperature may change the crystal form of mesoporous silica, and then lead to the destruction of the mesoporous structure. At the same time, the higher calcination temperature may also lead to the formation of Si — O — Si bonds due to the condensation of Si-OH groups on the surface of MCM-41 MSNs, thus reducing the density of Si-OH groups on the surface of MCM-41 MSNs, and then affecting the physical and chemical properties of the surface. In addition, high temperature calcination may also oxidize the template material in the mesoporous silicon channels into smelly or toxic gases. In order to improve the effect of removing template material by high temperature calcination, some scientists have proposed some improved methods, such as strengthening the network structure before calcination or using two-step calcination method
[90][91].
3.2 Solvent extraction method
Solvent extraction is a relatively mild method to remove the templating agent. Usually, the extraction solution (such as HCI alcohol solution) is used to clean the mesoporous silica particles containing surfactant template to remove the templating agent. The method is suitable for removing templates of various mesoporous silicon particles, and has an excellent effect on removing a template agent of the mesoporous silicon containing organic functional groups prepared by a co-condensation method; and meanwhile, the template agent can be well prevented from being decomposed in the process of removing the template agent, so that the template agent can be well recycled for repeated use. However, the removal efficiency of this method is low, and it generally needs repeated cleaning to achieve the desired removal effect. Usually, the removal effect of the template agent in the mesoporous silicon channel can be detected by a Fourier transform infrared spectrometer, for example, the removal condition of the template agent can be judged by the existence of a C-H characteristic peak of a 2800~3000 cm-1. At present, the commonly used solvent extraction methods can be divided into chemical extraction and supercritical fluid extraction.
The chemical extraction method is based on the dissolution of the template in the organic solvent. The MCM-41 MSNs containing the template is washed repeatedly in the organic solvent, and the template is separated from its pores to obtain the ordered MCM-41 MSNs. The method does not need high temperature, the mesoporous structure of the MCM-41 MSNs is not damaged in the template elution process, and the separated template can be recycled after certain treatment. However, the removal rate of this method is slow and time-consuming, and repeated cleaning is needed to achieve the desired effect of template removal. In general, different extraction systems have different elution temperature and time.
Du et al. mixed the synthesized MCM-41 MSNs with a hydrochloric acid-ethanol system (1 mL concentrated HCl: 100 mL ethanol) and magnetically stirred at room temperature to remove the templating agent
[92]. Liu et al. removed the template CTAB by immersing the synthesized MCM-41 MSNs/indium tin oxide electrode in 0.1 mol/L hydrochloric acid-ethanol solution and stirring at room temperature for 15 min
[93]. Xia et al. extracted the template CTAB from the prepared MCM-41 MSNs and hydrochloric acid-methanol solution in a reflux device, and the expected template removal effect was achieved after 24 H
[94].
Supercritical fluid extraction (SFE) is a method to obtain ordered MCM-41 MSNs by efficiently eluting the template from the pore channels of MCM-41 MSNs through the efficient dissolution of the template and supercritical fluid. The method has good template removal effect. However, this method requires specific instruments and strict experimental conditions, so its operation is limited.
Kawi extracted the MCM-41 MSNs with supercritical methanol -CO
2 at 85 ℃ and 35 MPa, and the extraction rate reached more than 90% within 3 H
[95]. Huang et al. Used methanol -CO
2 mixture as a supercritical fluid to extract the template in the channels of MCM-41 MSNs at 90 ℃ and 35 MPa, and achieved considerable extraction results
[96].
3.3 Microwave removal method
The microwave removal method is that a strong acid-strong oxidant solution is mixed with MCM-41 MSNs containing a template agent, and then the MCM-41 MSNs are subjected to microwave heating treatment. The template agent in the channels of MCM-41 MSNs can be oxidized and decomposed by the heat generated by microwave heating to achieve the effect of template removal. The method has the advantages of fast template removal rate, no need of high temperature, little influence on the mesoporous structure after template elution, and high degree of silanol (Si-OH) retention. However, this method must rely on expensive microwave instruments, and microwave radiation may also have a certain impact on the human body in the experimental operation. Zhao et al. Mixed a certain proportion of HNO
3-H
2O
2 mixture with MCM-41 MSNs containing the template in a polytetrafluoroethylene tube, and then heated it for 2 ~ 5 min under the radiation of a microwave instrument to efficiently remove the template in the mesoporous silicon channels
[83].
3.4 Ultrasonic treatment method
The ultrasonic treatment method is usually to mix the MCM-41 MSNs containing template with organic solvent (ethanol or methanol), and then carry out ultrasonic radiation treatment at a certain temperature and stirring rate to achieve the effect of template removal. The MCM-41 MSNs prepared by the method have higher specific surface area, larger pore volume and shorter template removal time than those prepared by the traditional high-temperature calcination method. In addition, the separated template agent can be recycled. Jabariyan et al. mixed the synthesized MCM-41 MSNs with ethanol or methanol and placed them in an ultrasonic instrument, and then carried out efficient template removal under 600 W ultrasonic frequency, 800 W thermal power, 28 KHz frequency and 3000 R/min rotation speed
[84].
3.5 Oxidation method
The oxidation method is a method to remove the template by decomposing the template into small molecules such as CO
2 and H
2O through appropriate redox reactions under certain conditions. The method has the advantages of mild conditions, no need of high temperature and ideal template removal effect. At present, there are two main methods to remove the template in the pores of MCM-41 MSNs by oxidation, namely ozone and H
2O
2 treatment
[97][98].
Ozone method is a method to remove template agent by oxidation-reduction reaction between ozone (O
3) and template agent under certain conditions, so that the template agent is decomposed into small molecules such as CO
2 and H
2O. According to the production mode of O
3, the ozone method at present can be divided into "ultraviolet-ozone method" and "ozone generator-ozone method". Keene et al. irradiated the dried MCM-41 MSNs containing the template with an ultraviolet lamp (ultraviolet power: 6.8 W, radiation wavelength: 254 and 180 nm) for 24 H, and then the generated O
3 oxidized and decomposed the template into small molecules such as CO
2 and H
2O
[99]. However, this method usually requires a long time and the amount of ozone generated cannot be adjusted. Based on the consideration of the above shortcomings, B Büchel et al. replaced the traditional ultraviolet lamp for generating O
3 molecules with an ozone generator with controllable ozone production, and introduced the O
3 molecules generated by the ozone generator into the aqueous solution of MCM-41 MSNs, and the template was efficiently removed after 30 min
[100].
The H
2O
2 treatment method is a method that the H
2O
2 is mixed with MCM-41 MSNs containing a template agent under certain conditions, and the template of the MCM-41 MSNs pore channel is decomposed into small molecules such as CO
2 and H
2O through a redox reaction to remove the template. The method has the advantages of mild reaction conditions, short reaction time and ideal template removal effect. Bein et al. Added 30% H
2O
2 into the pure water solution of MCM-41 mesoporous silica, and after refluxing at 95 ℃, the template agent in the mesoporous silica channels was well removed
[98]. The method has higher extraction efficiency than the traditional extraction method, can well control the size and the shape of the MCM-41 MSNs, and can not generate the phenomenon of irreversible particle agglomeration.
3.6 Other
In order to meet the considerable template removal effect under different experimental conditions, various novel template removal methods have been developed
[101⇓~103]. For example, Ariapad et al. Eluted the cationic surfactant template filled in the channels of MCM-41 MSNs by ion exchange method: first, MCM-41 MSNs containing cationic template was dispersed in NH
4NO
3- ethanol system and stirred for 15 min at 60 ℃
[104]; Then the solid particles are filtered and recovered, and the recovered solid is washed with cold ethanol; It was then dried again at 45.d egree. C. and the entire extraction procedure described above was repeated. The method can effectively remove the cation template in the channels of MCM-41 MSNs, not only does not destroy the mesoporous structure in the extraction process, but also makes the mesoporous channel walls of MCM-41 MSNs highly uniform and functionalized. For another example, Goworek et al. effectively removed the template by heating MCM-41 MSNs containing the template at a constant temperature of hydrogen flow (250 ℃) for 15 H
[105]. Compared with the traditional high temperature calcination method, this method not only does not produce pure carbon residues, but also does not destroy the mesoporous structure of MCM-41 MSNs. However, this method needs to strictly control the concentration of hydrogen to avoid explosion.
4 Surface Modification of MCM-41 Mesoporous Silica Nanoparticles
Although there are silicon dioxide tetrahedra terminated by siloxane (Si — O — Si) and silanol (Si — OH) on the framework surface of MCM-41 MSNs, and both of them can be functionalized, it is generally believed that the latter (silanol hydroxyl) is the main way to functionalize MCM-41 MSNs, that is, the surface functionalization of MCM- 41 MSNs is generally achieved through the covalent interaction between Si — OH on the surface of MCM- 41 MSN and functional molecules (such as organosilane)
[106]. The functionalized MCM-41 MSNs can utilize the functional groups at the mouth of the pore to combine with targeted molecules, so they have considerable application prospects in the field of biosensing.
4.1 Surface functionalized modification
The surface functionalization of MCM-41 MSNs is generally achieved by introducing target functional groups (such as amino, azido, thiol, carboxyl and sulfonic groups) on the surface of MCM-41 MSNs
[107⇓~109][78,110,111][112][113][114]. At present, there are two main strategies for surface functionalization of MCM-41 MSNs, namely, post-grafting and co-condensation
[106].
Post-grafting, also known as stepwise synthesis, is generally divided into two steps, namely, the synthesis of MCM-41 MSNs and the introduction of functional reagents. At present, there are three methods to introduce functional groups on the surface of MCM-41 MSNs by post-grafting, namely impregnation, ion exchange of surfactant and immobilization, among which immobilization is the most commonly used method
[115,116][117][118]. The immobilization method based on the post-grafting method is generally to prepare functionalized MCM-41 MSNs by reacting the synthesized MCM-41 MSNs with a functionalized silane coupling agent. Talavera-Pech et al. prepared amino-functionalized spherical MCM-41 MSNs by adding the synthesized spherical MCM-41 MSNs to a solution containing toluene and 3-aminopropyltriethoxysilane (APTES) and refluxing for 6 H
[119]. Khorshidi et al. added the dichloromethane solution of chlorosulfonic acid dropwise to the pre-synthesized Fe
3O
4@MCM-41 mesoporous silicon composite, and then prepared the sulfonated core-shell spherical MCM-41 MSNs after stirring, cleaning and vacuum drying
[120]. Sahoo et al. added the synthesized spherical MCM-41 MSNs with hollow structure to a solution containing toluene and APTES, and then stirred vigorously at 80 ℃ for 24 H. After washing, centrifugation and vacuum drying, amino-functionalized hollow spherical MCM-41 MSNs were prepared
[121].
The post-grafting method can effectively functionalize the surface of MCM-41 MSNs, and the original mesoporous structure and properties of MCM-41 MSNs are basically not destroyed after the introduction of functional groups. However, the inner and outer surfaces of MCM-41 MSNs are not selectively functionalized by this method, and more functional groups are usually introduced at the pore mouth and the outer surface. In addition, this method may cause the blockage of the channels because the molecules of the silane coupling agent are too large.
Co-condensation, also known as one-step synthesis, was first proposed by Burkett et al. Silicon source, functionalization agent (such as organosilane or metal ions), surfactant template and catalyst are usually added to the reaction medium simultaneously or sequentially.Surface-functionalized MCM-41 MSNs are formed around the surfactant template micelles by hydrolysis, intermolecular forces, and/or electrostatic forces, and these functional groups are uniformly distributed on the inner/outer surfaces of the mesoporous matrix
[122]. Vunain et al. added silicon source TEOS and organosilane APTES to the precursor solution containing CTAB and ammonia water in turn, and after stirring, filtering, template removal and drying, prepared the amino-functionalized spherical MCM-41 MSNs
[123]. Saadatjoo et al. prepared mercapto-functionalized core-shell spherical MCM-41 MSNs by mixing the synthesized Fe
3O
4 nanoparticles with ammonia, TEOS, NaOH, and CTAB, followed by hydrothermal treatment, template elution, and drying
[124].
The co-condensation method can synthesize MCM-41 MSNs and functionalize the materials at the same time. In addition, the functionalized groups are uniformly distributed on the inner/outer surfaces of MCM-41 MSNs and do not block the mesoporous channels. However, the mesoporous structure and physicochemical properties of MCM-41 MSNs may be affected by the addition of functionalized reagents to the precursor solution. In addition, the functionalized MCM-41 MSNs synthesized by this method can not use the high temperature calcination method for template removal (high temperature may destroy part of the organic functional groups).
4.2 Surface targeted modification
The functionalized MCM-41 MSNs can be further modified with other substances (such as metal nanoparticles or DNA molecules) through chemical bonds to meet the needs in the sensing field
[125⇓⇓~128][129]. For example, AuNPs or DNA molecules are connected to the pore surface of MCM-41 MSNs through chemical bonds, which can effectively improve the sensitivity of DNA biosensing technology
[130][131]. Zhu et al. constructed a highly selective "gating" system by interacting nucleated gold nanoparticles (AuNPs) with aptamer-modified mesoporous silica microspheres
[132]. Under the stimulation of the target aptamer, the system will break the AuNPs as a "gate molecule" and release the signal probe (glucose) originally loaded in the microsphere material. The released glucose molecules will show their specific content with the help of personal blood glucose meter, which can effectively detect the target aptamer accurately. Pascual et al. constructed a highly selective "oligonucleotide gating system" by combining mycoplasma aptamers with functionalized spherical MSNs
[133]. In this system, a single strand of DNA in the DNA duplex is replaced in the presence of mycoplasma DNA to cleave the duplex DNA previously immobilized on the microsphere. At this point, the "gating" system will release the signal indicator molecule, causing a change in the detected signal. The target (mycoplasma) was efficiently detected by the change of signal before and after the reaction. Roushani et al. constructed a high-sensitivity and high-selectivity streptomycin detection platform by integrating streptomycin aptamers and thin film MSNs immobilized with AgNPs at the orifice on an Au electrode
[134]. The immobilization of AgNPs makes the modified electrode have larger specific surface area and better conductivity, and at this time, the modified electrode will immobilize more streptomycin aptamers, so that the free diffusion of the electroactive probe Fe(CN)
63-/4- at the orifice of the film is hindered, the electrical signal is significantly reduced, and the generated strong signal difference can be used for efficient detection of streptomycin.
5 Application of MCM-41 Mesoporous Silica Nanoparticles in DNA Biosensor
The high efficiency immobilization of DNA probe and the high efficiency output of DNA hybridization signal are effective methods to improve the sensitivity of DNA biosensor
[135][136]. MCM-41 MSNs have been widely used to improve the sensitivity of DNA biosensors in recent years due to their excellent biocompatibility, stable chemical properties, large pore volume, adjustable pore size, large specific surface area and easy surface modification
[137]. At present, DNA biosensors based on MCM-41 MSNs mainly improve the sensitivity of detection through three functions of MCM-41 MSNs, namely, as a delivery system of signal probes, molecular sieves and carriers of active nanomaterials.
5.1 Delivery system as signal probe
MCM-41 MSNs have the ordered mesoporous structure with tunable pore size, high specific surface area and easy surface modification, which can load a large number of guest substances
[138]. Mesoporous silica loaded with a large number of guest substances can immobilize target response substances (such as heavy metal ions and biomolecules) at its pore mouth, which constitutes a kind of guest substance delivery system with gating effect
[139][131]. In the field of DNA biosensing, MCM-41 MSNs can construct a signal probe delivery system with gating effect by loading different signal probes (such as electrochemical probes and optical probes) and immobilizing DNA molecules with targeted response at its orifice
[140][141]. Under the effective stimulation of the target substance, the molecular gate at the orifice of the delivery system will be opened, releasing a large number of signal probes, which greatly increases the detection signal of various DNA biosensors, such as electrochemical DNA biosensors, fluorescent DNA biosensor, luminescent DNA biosensor and Photoelectrochemical DNA biosensor
[142][143][144,145][146].
5.1.1 Lectrochemical DNA biosensor
Electrochemical DNA biosensor has the advantages of low cost, strong recognition ability, high sensitivity, rapid response and good real-time detection
[147]. In the traditional label-free electrochemical DNA biosensor, the electrical signal is analyzed by detecting the binding between the electroactive indicator and the DNA probe before and after, and then the target substance is effectively detected
[148]. However, these methods often have some background interference signals, so they have some limitations in the efficient detection of target substances. The MCM-41 MSNs are capable of loading a large number of electroactive indicators
[149]. In the electrochemical DNA biosensor based on signal probe delivery system, the effective stimulation of the target substance will release a large number of electroactive indicators from the pore of MCM-41 MSNs
[150]. On the one hand, the controlled release of the electroactive indicator can greatly reduce the background interference signal during the detection; On the other hand, a large number of released electroactive indicators can greatly increase the electrical response signal and greatly improve the sensitivity of electrochemical DNA biosensors.
Wang et al. Prepared spherical MCM-41 MSNs with a pore size of about 2.5 nm and a particle size of 80 ~ 100 nm by hydrothermal method, and attached DNA single-stranded molecules rich in thymine, which can specifically bind to Hg
2+, to its surface by post-grafting method.The pores of MCM-41 MSNs are effectively blocked by a T-Hg
2+-T complex formed by the specific combination of Hg
2+ and the DNA single-stranded molecule, and an electroactive probe controlled release system specifically responding to GSH is constructed by utilizing the characteristic that glutathione (GSH) can be competitively combined with the Hg
2+ to enable the T-Hg
2+-T complex to be replaced into the DNA single-stranded molecule
[151]. In the presence of the target substance, the Hg
2+ undergoes a competitive displacement reaction, and the double-stranded DNA molecule ”T-Hg
2+-T complex is deformed into a single-stranded DNA molecule, thereby opening a pore valve and releasing a large amount of methylene blue, so that the detection signal is greatly improved.
Wen et al. Prepared spherical MCM-41 MSNs with a pore size of 2 ~ 3 nm and a particle size of about 100 nm by sol-gel method, and modified the surface of MCM-41 MSNs with amino groups by post-grafting method.The thiol-modified DNAzyme, which can be specifically cleaved by UO
22+, was immobilized on the outer surface of MCM-41 MSNs by covalent bonding with the help of a crosslinking agent, sodium imide ester, to prevent the release of methylene blue
[152]. Meanwhile, by utilizing the characteristic that the UO
22+ can specifically cut the DNA zyme molecule of the uranium mill into a DNA single chain, a pore valve is opened to release a large amount of methylene blue, so that an electric signal is greatly increased, and the detection sensitivity is effectively improved.
Cheng et al. Prepared spherical MCM-41 MSNs with a pore size of about 2.34 nm and a particle size of about 80 nm by sol-gel method, and loaded methylene blue on the surface of MCM-41 MSNs with azido by post-grafting method, and then anchored DNA molecules at its pore through the click chemistry of azido.An anchor DNA molecule is combined with a lambda phage exonuclease (lambda Exo) functional DNA and a target DNA according to a base pairing principle to construct an electrochemical DNA biosensing platform with a gating effect and low background cascade signal amplification, and the electrochemical DNA biosensing platform is used for detecting tumor-related mRNA with high sensitivity and high specificity
[153]. On this platform, the 5 '-phospho-terminal DNA(5'-PO
4cDNA) of the target mRNA was hybridized with the trigger DNA and anchor DNA at the pore of spherical MCM-41 MSNs to form a double-stranded DNA molecule with "gating effect" to block the methylene blue originally loaded in the pore. The 5'-PO
4cDNA will then competitively hybridize to the target mRNA and trigger cleavage of the double-stranded DNA and opening of the pore in the presence of lambda Exo. The released trigger DNA can be immobilized on the electrode surface by hybridization with the captured DNA, triggering the hybridization chain reaction (HCR) on the electrode surface. Meanwhile, the methylene blue released from the pores of spherical MCM-41 MSNs will be selectively inserted into the HCR long double-stranded DNA polymer, resulting in a significant electrochemical response. Finally, the tumor-associated mRNA was efficiently detected by the significant electrochemical signal changes before and after the detection.
5.1.2 Fluorescent DNA biosensor
The fluorescent DNA biosensor has the advantages of high sensitivity, good selectivity, obvious and stable detection effect and the like
[154]. Traditional fluorescent DNA biosensors have some limitations in high detection sensitivity because of the high cost of the label and the label may affect the activity of DNA molecules
[155]. MCM-41 MSNs can be loaded with a large number of fluorescent indicators. At the same time, the modification of DNA molecules at its orifice can make it form a target-selective controlled release system
[156]. Unlabeled fluorescent DNA biosensor based on MCM-41 MSNs Under the stimulation of the target substance, the fluorescent indicator in the pore of MCM-41 MSNs will be released in a large amount, which greatly increases the detection sensitivity
[157,158].
Dehghani et al. Prepared spherical MCM-41 MSNs with a pore size of 2 ~ 3 nm and a particle size of about 60 nm by a room temperature method, functionalized their amino groups by a post-grafting method, and then covalently immobilized DNA single strands for the formation of gating molecules.Aft that fluorescent probe rhodamine B is loaded, the kalamycin aptamer is fix according to the principle of base complementation, and the kalamycin can be competitively combined with the kalamycin aptamer, so that a DNA double strand is broken into a DNA single strand, and a large amount of rhodamine B is released at the moment, so that the fluorescence intensity is greatly increased, and the detection sensitivity is effectively improved
[159].
Oroval et al. Prepared spherical MCM-41 MSNs with a pore size of 2 ~ 3 nm and a particle size of about 100 nm by sol-gel method, and loaded the fluorescent probe Rhodamine B in the pore
[160]. Then the outer surface was modified by 3-aminopropyltriethoxysilane (APTES), and the positively charged amino moiety was bound to the negatively charged thrombin aptamer (TBA) by electrostatic interaction at neutral pH to construct a TBA blocked fluorescent probe release system. Thrombin can competitively combine with TBA to open the orifice valve and release a large amount of rhodamine B, which greatly increases the fluorescence signal and effectively improves the sensitivity of detection. Based on the principle of arsenic detection, the experimental group blocked the arsenic (III) aptamer in the orifice of spherical MCM-41 MSNs containing rhodamine B through electrostatic action, and carried out high-sensitivity detection of arsenic (III) (Fig. 1)
[161].
5.1.3 Chemiluminescent DNA biosensor
Chemiluminescent DNA biosensor is one of the most popular biosensors recently, which can detect the target substance by detecting the light intensity produced by the chemical reaction. It has the advantages of low background interference signal, simple operation, fast analysis speed, low cost and high sensitivity
[162]. This kind of sensor is generally based on the typical chemiluminescence system (luminol-H
2O
2 luminescence system), and combined with DNA molecules with the help of other nano-materials, which solves the problem that the traditional chemiluminescence method can only detect substances directly related to its luminescence system (such as H
2O
2)
[163]. MCM-41 MSNs can well combine DNA molecules with traditional chemiluminescence systems. Chemiluminescent DNA biosensor based on MCM-41 MSNs can not only solve the problem of single detection substance in traditional chemiluminescent biosensor, but also greatly increase the chemiluminescence signal output through the controlled release of the chemiluminescence catalyst substance in the channel of MCM-41 MSNs, which greatly improves the detection sensitivity
[144,164].
Chen et al. Prepared spherical MCM-41 MSNs with a pore size of about 2.2 nm and an average particle size of 150 nm by a room temperature method, functionalized the surface of MCM-41 MSNs with amino groups by a post-grafting method, and loaded glucose molecules which can promote the reaction of luminol chemiluminescence system.The electrostatic interaction between the positively charged amino group and the negatively charged cocaine aptamer was used to selectively block the orifice of MCM-41 MSNs, and a controlled release system of cocaine aptamer as a gate molecule of chemiluminescence inducer was constructed
[164]. In this system, cocaine can competitively bind to its aptamer, opening the orifice valve and releasing a large number of glucose molecules. At the same time, glucose reacts with dissolved oxygen to produce hydrogen peroxide with the assistance of glucose oxidase (GOx), which greatly increases the chemiluminescence signal of luminol system and greatly improves the sensitivity of detection.
Sun et al. Prepared spherical MCM-41 MSNs with a pore size of 2 ~ 3 nm and a particle size of about 60 nm by sol-gel method. After modifying amino groups on the surface of MCM-41 MSNs by post-grafting method, the heme, a chemiluminescent inducer, was encapsulated by MCM-41MSNs, and then the thrombin aptamer was sealed in the orifice by electrostatic interaction to construct an effective chemiluminescent probe controlled release system
[144]. By that stimulation of the thrombin modify with magnetic particles, the thrombin aptamer can competitively combine with the thrombin, so that the gate control molecule of the orifice is separated from the outer surface, a large amount of heme is released, and the chemiluminescence reaction of a luminol-H
2O
2 system is effectively catalyze. Meanwhile, the thrombin modified by the magnetic nanoparticles can be separated by a magnet for repeated use, thereby greatly improving the sensitivity and reproducibility of detection.
5.1.4 Lectrochemiluminescence DNA biosensor
Electrochemiluminescence DNA biosensor is a kind of DNA biosensor combining electrochemistry and chemiluminescence, which has the advantages of low background signal, high sensitivity, high selectivity and convenient detection
[165]. Among the types of electrochemiluminescence DNA biosensors, label-free electrochemiluminescence DNA biosensors have the advantages of convenient detection, low cost, high sensitivity and good selectivity
[166]. However, the traditional label-free electrochemiluminescence DNA biosensor is generally a "signal off" structure, which reduces the intensity of electrochemiluminescence and the sensitivity of detection. Therefore, this type of sensor requires further signal amplification. MCM-41 MSNs can be loaded with a large number of electrochemical luminophores. At the same time, DNA molecules are modified at the orifice thereof to constitute an electrochemiluminescent delivery system having controlled release properties. Under the stimulation of the target substance, the delivery system releases a large amount of electrochemiluminescence, which greatly increases the intensity of electrochemiluminescence and greatly improves the sensitivity of sensing
[167].
Yang et al. Prepared amino-functionalized magnetic spherical MCM-41 MSNs with a pore size of 2 ~ 3 nm and a particle size of about 100 nm through a hydrothermal method combined with a co-condensation method. After loading an electrochemical optical probe [Ru(bpy)
3]
2+, negatively charged thrombin aptamers were adsorbed on the surface of positively charged mesoporous silica spheres by electrostatic interaction to construct an effective ECL probe controlled release system
[145]. In the presence of the target substance thrombin, the thrombin aptamer will combine with it to form a more stable G-quadruplex structure.
Wang et al. Prepared spherical MCM-41 MSNs with a pore size of about 2. 4 nm and a particle size of about 100 nm by sol-gel method, and functionalized the surface of MCM-41 MSNs with amino groups by post-grafting method, and loaded with [Ru(bpy)
3]
2+.An electrochemiluminescence (ECL) probe delivery system controlled by survivin mRNA was constructed by immobilizing a single-stranded DNA molecule, which can pair with survivin mRNA, on the surface of mesoporous silica spheres by electrostatic interaction and blocking the pore
[168]. Upon stimulation of survivin mRNA, the aptamer molecule will selectively hybridize to it. At the same time, the target substance survivin mRNA is recycled under the action of double-stranded specific nuclease (DSN), which greatly improves the sensitivity of detection.
In addition to the typical spherical MCM-41 MSNs loaded with [Ru(bipy)
3]
2+ to construct the ECL probe controlled release system, Liu et al also prepared thin film MCM-41 MSNs with pore size of 2 ~ 3 nm and thickness of about 90 nm on ITO electrode by modified St Stöber method.A simple, convenient and label-free electrochemiluminescence aptamer sensing platform was constructed by using APTES to modify the positively charged amino groups on the surface, and then the negatively charged target aptamer was adsorbed on the external surface by electrostatic interaction to block the orifice
[169]. On this platform, the aptamer can be effectively adsorbed on the surface of the aminated silica film through non-covalent electrostatic adsorption, and can be used as an ideal gate material to effectively block and release the luminescent reagent Ru(bipy)
32+ adsorbed in the pore channel of the film. In the presence of the target substance, the specific aptamer-target binding can trigger the "gating" effect to release Ru(bipy)
32+, and the ECL signal will be significantly reduced. Further, the target substance is efficiently detected by the signal change before and after the reaction (fig. 2).
5.1.5 Photoelectrochemical DNA biosensor
Photoelectrochemical DNA biosensor is a new type of biosensor, which has the advantages of simple equipment, low background interference, short response time and high selectivity
[170]. However, the conjugation effect of DNA molecules and photoactive materials will not only affect the photoelectric efficiency and stability of photoactive materials, but also affect the biological activity of DNA molecules in this kind of sensors. Therefore, such sensors generally require photoactive materials with high photoelectric efficiency and high stability and signal amplification to improve their sensitivity
[171][172][173]. Signal amplification is one of the most commonly used methods
[174]. MCM-41 MSNs can be loaded with a large amount of Photoelectrochemical indicators, which can be released in large quantities in the presence of target substances. At this time, the Photoelectrochemical detection signal will be greatly increased, which greatly improves the sensitivity of this kind of sensor.
Zhou et al. prepared spherical MCM-41 MSNs with pore size of 1.83 ~ 2.81 nm and particle size of about 60 nm by sol-gel method, modified amino groups on its surface by post-grafting method, transferred glucose molecules as electron donors, and immobilized double-stranded DNA of biological gating molecules at its orifice by covalent interaction to construct a controlled release system for amplifying FeOOH-rGO photocurrent
[175]. In the presence of prostate specific antigen (PSA), the biomolecular gate is opened to release a large number of glucose molecules, which greatly increases the photocurrent and effectively improves the detection sensitivity. In addition, a new electrochemiluminescence detection platform for carcinoembryonic antigen was constructed by using bismuth iron oxide (BiFeO
3) nanostructures as photoactive materials and the target release control system mentioned above
[146]. On this platform, based on the oxidation of glucose by glucose oxidase, the produced enzyme product (H
2O
2) was used as an electron acceptor to increase the photocurrent of BiFeO
3 nanostructures under UV-visible light irradiation, and the highly sensitive detection of low concentration target carcinoembryonic antigen was realized.
5.1.6 Other types of DNA biosensors
Although the traditional types of DNA biosensors have the advantages of high sensitivity and good specificity, these sensors have some limitations, such as the need for expensive instruments, complex operation, high detection requirements and complex cleaning procedures
[176]. In addition, in practical detection situations, it is sometimes necessary to detect in time and make the sensor portable. As a result, these traditional types of DNA biosensors will no longer meet the demand for instant portable detection. Personal blood glucose meter (PGM) is one of the most widely used instant portable detectors because of its small size, easy operation, low cost and reliable quantitative results
[177]. However, traditional personal blood glucose meters usually require special enzyme labels, which require sample separation and multi-step cleaning operations, making their detection steps complex
[178]. MCM-41 MSNs can be loaded with a large number of glucose molecules. Aft a large amount of glucose molecules are fil in that pore canal and DNA molecules are modified on the pore opening, the pore canal can be well combine with a personal blood glucose meter to form a novel personal blood glucose meter. This new personal blood glucose meter based on MCM-41 MSNs overcomes the shortcomings of traditional DNA biosensors in terms of portability and detection timeliness, and has the advantages of low cost, rapid detection, high sensitivity, good selectivity and wide application range, so it is a very commercial sensor
[179].
Liang et al. prepared spherical MCM-41 MSNs with a pore size of about 2. 7 nm and a particle size of about 150 nm by a sol-gel method, modified the surface of the MCM-41 MSNs with positively charged amino groups by a post-grafting method, loaded glucose molecules, and wrapped single-stranded DNA molecules containing T-Hg
2+-T sequences in the orifice by electrostatic interaction to construct a signal probe release system with controlled release effect on Hg
2+[180]. With the help of helper DNA, Hg
2+ competitively binds to the gating molecule (single-stranded DNA) and releases a large number of signaling molecules (glucose), which effectively improves the detection signal of PGM.
Hou et al. Prepared spherical MCM-41 MSNs with a pore size of about 3.2 nm and a particle size of about 120 nm by sol-gel method, modified them with glutaraldehyde, immobilized the single chain of aminated DNA1 on their pores by covalent interaction, and loaded the signal probe glucose.The DNA 2 single-stranded molecule modified by the AuNPs and containing an ATP aptamer sequence is paired and combined with the DNA 1 single-stranded molecule, so that the AuNPs is separated from the orifice, a large number of glucose molecules are released, the reading of the PGM is greatly improved, and the detection sensitivity is effectively improved
[132].
5.2 As a molecular sieves
Among the MCM-41 MSNs, the film-shaped MCM-41 MSNs are ideal sieves for controlling particle transport
[181]. As "molecular sieves", thin film MCM-41 MSNs can be selectively transported by the basic properties of the particles, such as particle size, electrostatic and lipophilic properties
[182][183][184]. In the field of DNA biosensing, it is usually used as a modified layer of the electrode to combine with DNA molecules, so that the electroactive indicator can be selectively transported to the electrode surface by using the size of the particle to achieve efficient detection of target substances, such as in electrochemical DNA biosensors and electrochemiluminescence DNA biosensors
[77][183].
Electrochemical DNA biosensor has the advantages of low cost, high recognition ability, high sensitivity and real-time detection, and has a wide range of applications in the actual detection of biological samples
[185]. The traditional label-free electrochemical DNA biosensor usually immobilizes DNA molecules on the surface of the electrode, and detects the target substance by using the magnitude of the electrical signal generated when the electroactive probe is attached to the DNA molecules before and after detection
[186]. In addition, the actual sample is generally pretreated before detection to prevent organic matter, biological macromolecules, microorganisms and other impurities from adsorbing on the surface of the electrode and affecting the stability of the electrical active area and electrical response. However, the pretreatment of the sample usually requires a series of complex and time-consuming steps, and the detection will be accompanied by a certain background interference signal, which makes the electrochemical DNA biosensor have certain limitations. The thin film MCM-41 MSNs can be used as an effective anti-fouling layer material. After the film is fixed on the surface of the electrode, a composite electrode with better performance can be formed. The composite electrode can effectively prevent the interference of non-target impurities on the activity of the electrode, and can also omit the problem of sample pretreatment in the traditional detection process, so that the electroactive probe can be efficiently transmitted to the surface of the electrode, and the sensitivity and practicability of the electrochemical DNA sensor are greatly improved
[187].
Saadaoui et al. Prepared a thin film of MCM-41 MSNs with a pore size of about 2.5 nm and an average thickness of about 46 nm on a gold electrode by an electrochemical-assisted method, modified the pore mouth with an epoxyethyl group by a post-grafting method, and then combined with an amino-modified Cu
2+DNAzyme in the presence of a Cu
2+ to seal the pore of the film, thus constructing a simple and convenient label-free electrochemical DNA biosensing platform
[57]. On this platform, the Cu
2+DNAzyme acts as a "gate molecule" switch, which can effectively prevent the Fe(CN)
63-/4- of electrochemical probes from diffusing to the electrode surface and effectively reduce the background signal of detection. At the same time, in the presence of ascorbic acid, the Cu
2+DNAzyme will be decomposed into DNA single strands, at this time, the molecular gate is opened, and a large number of Fe(CN)
63-/4- diffuse to the surface of the electrode, greatly increasing the electrochemical signal.
Argoubi et al. prepared a mesoporous silicon film with a pore size of about 2 ~ 3 nm and a thickness of about 40 nm on a gold electrode by an electrochemical-assisted method, and immobilized a prostate-specific antigen (PSA) aptamer at the pore by covalent interaction after the surface of the film was modified with an epoxyethyl group, thus constructing a simple and convenient label-free electrochemical aptamer detection platform
[188]. On the platform, PSA aptamers can combine with PSA to form a stable and rigid complex with a certain size. At this time, the compound will block the orifice of the film, effectively prevent the Fe(CN)
63-/4- of the electroactive probe from diffusing to the surface of the electrode, and greatly reduce the electrochemical signal. The target substance PSA can be detected with high sensitivity through the change of electrochemical signals before and after the reaction.
In addition to the above common electroactive probe molecular sieves based on molecular size, Sun et al. Also constructed an electrochemical molecular sieve based on molecular lipophilicity
[184]. They prepared a mesoporous silicon film with a pore size of 2-3 nm and a thickness of about 86 nm on an ITO electrode by a modified St Stöber method, and constructed a simple and label-free electrochemical DNA sensing platform to detect 3,3 '-5,5' -tetramethylbenzidine (TMB). On this platform, the neutral/lipophilic compound TMB is preconcentrated by the lipophilic hexadecyl ammonium (CTA
+) in the pore of the thin film molecular sieve, so that TMB diffuses to the surface of the ITO electrode to cause electron transfer, and the response current for quantitative analysis is generated.
In addition, Xiong et al. prepared a mesoporous silica film containing sol, chitosan and [Ru(bpy)
3]
2+ on a graphite electrode by using an electrochemical-assisted method, adsorbed negatively charged DNA single strands on the surface of the positively charged film modified by chitosan by using electrostatic interaction, and constructed a label-free electrochemiluminescence platform based on the size screening and electrostatic effect of molecular sieves
[183]. First, the single-stranded DNA1 molecules are electrostatically adsorbed at the pore of the membrane, which greatly "reduces" the pore size, so that the oxalate ions (C
2O
42-), the co-reactant of ECL, can not diffuse into the pore of the membrane, and only a very weak ECL signal can be obtained from the platform. Subsequently, in the presence of the target DNA 2 single strand, the DNA 1 single strand previously adsorbed at the membrane orifice will competitively bind to it. When the orifice is opened, a large number of negatively charged oxalates (C
2O
42-) will diffuse into the pores of the film and combine with the positively charged [Ru(bpy)
3]
2+ on the inner wall of the pores through electrostatic interaction, which greatly increases the electrochemiluminescence signal.
5.3 As a carrier for active nanomaterials
MCM-41 MSNs can be used as a good carrier of active nanomaterials, such as noble metal nanoparticles and fluorescent quantum dots
[189][190]. The active nanoparticles immobilized on the carrier can not only improve the effect of their own, but also effectively avoid the adverse effects caused by their own aggregation
[191]. In the field of DNA biosensing, MCM-41 MSNs are usually used as carriers of active nanomaterials such as gold nanoparticles (AuNPs) and fluorescent quantum dots to effectively increase the sensing signal, such as in electrochemical DNA biosensors and fluorescent DNA biosensors
[192][193][194][195].
5.3.1 Lectrochemical DNA biosensor
The detection of electrical signals in electrochemical DNA biosensors is generally achieved by effective electrical communication between DNA molecules and electrodes
[196]. Therefore, improving the effective electrical communication between DNA molecules and electrodes is an effective way to improve the sensitivity of electrochemical DNA biosensors
[197]. In general, DNA molecules can be bound to the electrode through noble metal particles (such as AuNPs) with good conductivity and biocompatibility, which effectively improves the electrical communication between them and greatly increases the sensitivity of electrochemical DNA biosensors
[198]. However, in practice, noble metal nanoparticles such as AuNPs may aggregate and affect their performance of improving electron transfer. MCM-41 MSNs can immobilize a large number of noble metal nanoparticles such as AuNPs, so that AuNPs are orderly dispersed on MCM-41 MSNs. The MCM-41 MSNs immobilized with a large number of AuNPs and modified with DNA molecules can greatly improve the effective electron transfer between DNA molecules and the electrode, and greatly increase the sensitivity of sensing detection
[199].
Tang et al. Prepared spherical MCM-41 MSNs with a pore size of about 2. 3 nm and an average particle size of 150 nm by a room temperature method. After loading the electrical signal molecule thionine, AuNPs were used to seal the pore to construct a highly sensitive electrical signal amplification carrier (GMSNs)
[200]. In this carrier, AuNPs can not only effectively prevent the leakage of signal tag thionine molecules, but also have good conductivity, which greatly improves the performance of composite electrical signal tag GMSNs. In addition, the layered dendrite gold microstructure modified by thrombin aptamer 1 was immobilized on a glassy carbon electrode, and then it cooperated with GMSNs modified by thrombin aptamer 1 to detect thrombin with high sensitivity.
Roushani et al. immobilized a thin film with a pore size of 2 ~ 3 nm and a thickness of about 40 nm on the surface of a gold electrode by an electrochemical-assisted method, functionalized the surface with amino groups by a post-grafting method, immobilized AgNPs by covalent interaction, and immobilized streptomycin aptamer on the surface of the thin film by Ag — S bond to construct a carrier modified layer with large specific surface area, good conductivity and biocompatibility
[134]. The gold electrode fixed with the modified layer can bind more streptomycin aptamers. Meanwhile, the electric response between the aptamer and the electrode is greatly increased, and the detection sensitivity is effectively improved.
In addition to improving the electrochemical detection signal by immobilizing noble metal nanoparticles through MCM-41 MSNs, MCM-41 MSNs can also be combined with carbon nanotubes or/and graphene to improve the electrical detection signal. Zhang et al. Coated MCM-41 MSNs on multi-walled carbon nanotubes by hydrothermal method to make it a composite material with better surface modification and biocompatibility
[201]. An electroactive indicator thionine molecule is loaded in the composite material, and platinum nanoparticles, amino-modified thrombin aptamer, heme and bovine serum albumin are sequentially immobilized to construct a composite carrier for multiple electric signal amplification. In the presence of thrombin, the synergistic effect of the carrier and the dendrimer-functionalized redox graphene greatly promotes the transfer of electrons from thionine to the electrode and effectively improves the sensitivity of detection. Guo et al. Also combined graphene with AuNPs through MCM-41 MSNs to construct a new type of electrical signal increasing element
[202]. At the same time, the double signal amplification strategy of strand displacement DNA polymerase and parallel motif DNA triple stranded system was used to realize the ultra-sensitive detection of ATP molecules.
5.3.2 Fluorescent DNA biosensor
The fluorescent DNA biosensor has the advantages of high specificity, good sensitivity, obvious and stable detection effect and the like
[203]. In order to make fluorescent DNA biosensors have higher sensitivity, fluorescent probes with high fluorescence effect are usually used
[204]. Fluorescent quantum dots (QDs) have many advantages, such as large Stokes shift, high fluorescence efficiency, broad excitation spectrum, narrow emission spectrum, resistance to photobleaching, and the ability to excite multiple fluorescent colors simultaneously, which make them a good choice for high efficiency fluorescent probes
[205]. In addition, MCM-41 MSNs can immobilize various fluorescent dyes including fluorescent quantum dots
[206,207]. Combining fluorescent QDs with MCM-41 MSNs can construct an ideal and high-performance composite fluorescent probe. The composite fluorescent probe not only has a high-performance fluorescent effect, but also has the advantages of good biocompatibility, easy surface modification and the like, can greatly improve a fluorescent signal during sensing detection, and greatly increases the sensitivity of a fluorescent DNA biosensor
[193,195].
Liang et al. prepared MCM-41 MSNs with a pore size of about 8 ~ 9 nm and a particle size of about 100 nm by sol-gel method. After modifying amino groups by post-grafting method, a large number of quantum dots with good fluorescence effect were immobilized on the surface of MCM-41 MSNs to form a signal label with high fluorescence effect
[193]. After binding to the flexible aptamer molecule, the label can be adsorbed on the surface of graphene oxide (GO), and the target cells can be efficiently detected by comparing the fluorescence quenching caused by F Förster resonance energy transfer with the fluorescence recovery after adding to the target cells.
Based on the above detection system, Sun et al used MCM-41 MSNs loaded with a large number of fluorescent quantum dots as efficient fluorescent labels to label carcinoembryonic antigen (CEA) aptamers and prostate specific antigen (PSA) aptamers
[195]. The fluorescently labeled aptamers can be adsorbed on the surface of molybdenum disulfide, and the fluorescence quenching caused by F Förster resonance energy transfer and the fluorescence recovery after the addition of target substances (CEA and PSA) are contrasted to detect the target substances with high sensitivity.
5.3.3 Other DNA biosensors
In recent years, this kind of complex carrier with activity has also been well applied in new DNA biosensors. Zhu et al. Immobilized a large number of fluorescent quantum dots CdTe on the prepared MCM-41 MSNs to construct an efficient electrochemiluminescence signal probe
[208]. In addition, with the assistance of magnetic nanoparticles (Fe
3O
4-AuNPs) modified with AuNPs, magnets, and double-stranded specific nuclease (DSN), the detection of microRNA was carried out by double ECL signal amplification on the surface of a glassy carbon electrode. Ge et al. Constructed a new type of Photoelectrochemical signal probe by modifying CdTe fluorescent nanoparticles with MCM-41 MSNs (GMSNs) assembled with AuNPs
[209]. Under the dual signal amplification strategy of graphene quantum dots and multi-branched hybrid chains, the probe greatly increased the photocurrent efficiency and carried out ultrasensitive detection of breast cancer cells (MCF-7) and N-glycans on the cell surface (Fig. 3).
In addition, MCM-41 MSNs can also be combined with magnetic nanoparticles (Fe
3O
4) to make them magnetic. Magnetic MCM-41 MSNs can not only modify a large number of biomolecules such as DNA, but also realize the separation and enrichment of target substances in the detection process, which greatly improves the sensitivity of target substance detection
[210,211].
6 Conclusion and prospect
The application of MCM-41 MSNs in the field of DNA biosensing has attracted more and more attention because of its good biocompatibility, ordered mesoporous structure, adjustable pore size, large pore volume, large specific surface area, easy surface modification, controllable morphology, good stability and dispersibility. However, there are still some problems to be solved in its preparation and application. Firstly, in terms of raw materials, the price of silicon source and organic template for the preparation of MCM-41 MSNs is high, and the reagents constituting the precursor solution need to maintain a suitable parameter ratio. Therefore, it is necessary to develop templates with higher cost performance. Secondly, in terms of synthesis, the current synthesis methods generally require more operation steps, and the yield is low, the template is difficult to completely remove, which is not conducive to large-scale synthesis. In addition, the MCM-41 MSNs synthesized at this stage need to be further optimized in terms of product collection, resuspension, homogeneity and solubility. Therefore, there is a need to develop a method that can effectively reduce the hydrolysis and condensation time of the silicon source and efficiently remove the template agent. Finally, the isoelectric point of MCM-41 MSNs is 2, which may combine with positively charged indicator substances in solution and reduce the sensitivity of detection. Therefore, there is a need to develop more high-performance signal indicators.
Although DNA biosensors based on MCM-41 MSNs have attracted more and more attention from scientists at this stage, the research in this area is still in its infancy and still needs further optimization. Based on the extensive research at this stage, the following three aspects can be considered in the optimization scheme of DNA biosensors based on MCM-41 MSNs: First, explore more mesoporous structures and functions of MCM-41 MSNs, and expand the types of DNA biosensors to improve their sensitivity. Secondly, more MCM-41 MSNs with excellent shape and properties were synthesized to improve the sensitivity of DNA biosensors. Thirdly, MCM-41 MSNs are combined with other active nanomaterials and other signal amplification technologies to develop more efficient methods to improve the sensitivity of DNA biosensors. With the rapid development of nanotechnology based on MCM-41 MSNs and new DNA biosensing technology, DNA biosensors based on MCM-41 MSNs are expected to have a broader application prospect in the future DNA biosensing field.
National Natural Science Foundation of China(11475079)
Open Project Program of the State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University(2018010)
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