Emulsion-oriented assembly for Janus double-spherical mesoporous nanoparticles as biological logic gates

The ability of Janus nanoparticles to establish biological logic systems has been widely exploited, yet conventional non/uni-porous Janus nanoparticles are unable to fully mimic biological communications. Here we demonstrate an emulsion-oriented assembly approach for the fabrication of highly uniform Janus double-spherical MSN&mPDA (MSN, mesoporous silica nanoparticle; mPDA, mesoporous polydopamine) nanoparticles. The delicate Janus nanoparticle possesses a spherical MSN with a diameter of ~150 nm and an mPDA hemisphere with a diameter of ~120 nm. In addition, the mesopore size in the MSN compartment is tunable from ~3 to ~25 nm, while those in the mPDA compartments range from ~5 to ~50 nm. Due to the different chemical properties and mesopore sizes in the two compartments, we achieve selective loading of guests in different compartments, and successfully establish single-particle-level biological logic gates. The dual-mesoporous structure enables consecutive valve-opening and matter-releasing reactions within one single nanoparticle, facilitating the design of single-particle-level logic systems. Large biomolecules cannot be loaded into conventional Janus nanoparticles with small mesopores, preventing the establishment of efficient logic-gate systems in single Janus nanoparticles. Now, an emulsion-oriented assembly approach has been shown to fabricate Janus double-spherical nanoparticles with dual-tunable mesopores, enabling the design of various single-particle-level logic systems.

The ability of Janus nanoparticles to establish biological logic systems has been widely exploited, yet conventional non/uni-porous Janus nanoparticles are unable to fully mimic biological communications. Here we demonstrate an emulsion-oriented assembly approach for the fabrication of highly uniform Janus double-spherical MSN&mPDA (MSN, mesoporous silica nanoparticle; mPDA, mesoporous polydopamine) nanoparticles. The delicate Janus nanoparticle possesses a spherical MSN with a diameter of ~150 nm and an mPDA hemisphere with a diameter of ~120 nm. In addition, the mesopore size in the MSN compartment is tunable from ~3 to ~25 nm, while those in the mPDA compartments range from ~5 to ~50 nm. Due to the different chemical properties and mesopore sizes in the two compartments, we achieve selective loading of guests in different compartments, and successfully establish single-particle-level biological logic gates. The dual-mesoporous structure enables consecutive valve-opening and matter-releasing reactions within one single nanoparticle, facilitating the design of single-particle-level logic systems.
Mesoporous nanomaterials with large surface area, high pore volume and tunable pore sizes and mesostructures have been widely used in a number of fields 1 , including catalysis 2,3 , energy conversion and storage [4][5][6][7] , and biomedicine [8][9][10][11][12] . Mesoporous nanoparticles with unique morphologies and structures have been fabricated during recent decades, enabling great enhancements in properties that are useful for various applications [13][14][15][16] . However, single-compartment mesoporous nanoparticles fail to meet the demands of intricate applications such as biological logic gates and multi-model drug release, as they are unable to provide multiple storage spaces in a single nanoparticle.
Mesoporous Janus nanoparticles, because of their independent mesoporous compartments, spatially isolated surfaces and compositions, have attracted a great deal of attention [14][15][16][17][18][19] . A range of asymmetric mesoporous nanoparticles have been fabricated, including Janus 20 , multipod 21 , winding 19 , nanotruck 22 , di-block and tri-block 23 nanoparticles, and various advanced applications have been designed based on their separate mesoporous compartments, such as cascade catalysis 24,25 , multi-model drug delivery 20 and so on. So far, the fabrication of Janus mesoporous nanomaterials has been largely based on the anisotropic assembly of mesoporous compartments and requires the dual compartments of the Janus nanoparticles to have distinct mesostructures. Accordingly, most of the Janus nanoparticles reported previously contain at least one compartment with nanocube or nanorod morphology (for example, sphere-cube, sphere-rod or Article https://doi.org/10.1038/s41557-023-01183-4 Developing biological logic systems at the single-particle level thus calls for Janus mesoporous nanoparticles with novel architectures. We postulated that an emulsion-oriented assembly strategyin which oil droplets interact with mesoporous silica nanoparticles (MSNs) to form double-spherical structures, and subsequently direct the selective encapsulation of mesoporous polydopamine (mPDA) on the oil droplet-would lead to the formation of Janus double-spherical MSN&mPDA nanoparticles with large mesopores (Fig. 1a). We proposed that various enzymes, supramolecular nanovalves and model molecules could be selectively loaded into different compartments of the Janus MSN&mPDA nanoparticles, establishing YES, OR and AND biological logic gates (Fig. 1b,c). Furthermore, the dual-mesoporous structure enables consecutive valve-opening and matter-releasing reactions within one single nanoparticle, which will greatly expand the scope of the design and complexity of biological logic gates.

Characterization of the mesoporous Janus nanoparticles
Spherical MSNs with a uniform diameter of ~150 nm and a radial mesopore channel of ~8 nm were synthesized by means of a bi-phase method, using cetyltrimethylammonium bromide (CTAB) as a structure-directing agent ( Supplementary Fig. 1) 41 . After the extraction of CTAB from the MSNs using a solvent backflow method ( Supplementary Fig. 2), the Janus MSN&mPDA nanoparticles were formed through an emulsion-oriented assembly of 1,3,5-trimethylbenzene (TMB), pluronic F-127, CTAB and polydopamine (PDA) on the pre-made MSNs (Fig. 2a). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images show that one mPDA hemisphere with a diameter of ~120 nm and a radial mesopore channel are grown on each MSN, forming a double-spherical Janus MSN&mPDA nanoparticle (Fig. 2b,c). Large-scale TEM (Fig. 2d) and SEM ( Supplementary Fig. 3) images demonstrate that the obtained Janus nanoparticles are very uniformly constructed and well-dispersed. Statistical analysis of the morphologies of 200 randomly selected nanoparticles shows that nearly 80% of the obtained nanoparticles have the same double-spherical structure, indicating the controllability of the method ( Supplementary Fig. 4). Bowl-like mPDA hemispheres can be obtained after etching the MSN nanospheres, further verifying the morphology of the Janus MSN&mPDA nanoparticle ( Supplementary Fig. 5).
cube-rod structured Janus nanocomposites). It remains a great challenge to synthesis Janus mesoporous nanoparticles with identical mesostructured compartments, impeding the formation of diverse structures such as the peanut-like double-spherical architecture. Also, the specific mesostructure further restricts adjustment of the pore size, so the obtained Janus mesoporous nanoparticles have only small mesopores (<3 nm). The limited pore sizes of Janus mesoporous nanoparticles hinder their capacity to load large-sized functional guests such as bio-macromolecules, restricting their potential application. There is thus an urgent need to develop new strategies for the fabrication of Janus mesoporous nanoparticles with novel architectures and tunable pore sizes.
The ability to transport information via biomolecules is crucial in biology, with relevance to the behaviour of animals and cellular activities. Biological logic gates utilize information-processing biomolecules to establish internal logic systems, similar to those in electronic devices, therefore greatly expanding the potential applications of biomolecular engineering. Such logic gates are usually established based on large-sized functional bio-macromolecules, such as nucleic acids [26][27][28][29] , proteins/ enzymes 30,31 and supramolecular valves [32][33][34] . A range of biological logic gates have been built in the form of gels 31 , polymers 26 and tablets 35 , and these have recently advanced to the single-particle level based on Janus nanoparticles [36][37][38][39][40] . Despite previous successes, however, conventional biological logic gates, especially single-particle-level ones, rely on enzymatic reactions as an indispensable information processor. This is because these nanoparticles are mostly single-component, or multi-component nanoparticles but with only one porous subunit. They are unable to provide independent storage spaces for multiple intermediate and output signalling molecules. Thus, at least one processing signal requires enzymes to transform the inputs and avoid disruption of the ordered logic, which limits the scope and complexity of biological logic gates. Janus mesoporous nanoparticles with dual independent mesopores possess the ability to load multiple cargoes in different compartments. These dual-mesoporous Janus nanoparticles can effectively separate the input, internal-processing and output units, and provide many more possibilities for the construction of novel biological logic gates. However, single-particle biological logic gates built on small-pore Janus nanoparticles suffer from the low loading capacity of bio-macromolecules, with consequent inefficiency.   Different elements in the MSN domain (silicon) and mPDA hemisphere (carbon and nitrogen) can be identified by element mapping (Fig. 2e), further demonstrating the unique asymmetric double-spherical morphology.
Nitrogen sorption isotherms of the obtained dual-mesoporous MSN&mPDA Janus nanoparticles show two capillary condensation steps in the relative pressure P/P 0 ranges of 0.4-0.6 and 0.8-0.9, evidencing the coexistence of two sets of uniform mesopores (Supplementary Fig. 6) 42,43 . The Brunauer−Emmett−Teller surface area was measured to be ~243 m 2 g −1 with a high pore volume of ~0.56 cm 3 g −1 . The pore size distribution calculated using the Barrett-Joyner-Halenda (BJH) model demonstrates two sets of narrowly distributed mesopores at ∼7.8 and ∼14 nm, respectively (Fig. 2f), which matches the TEM observations well (Fig. 2g,h). TEM images of ultra-thin slices of the Janus nanoparticle demonstrate that the mesopores in the mPDA section are not interconnected with those in the MSN (Supplementary Fig. 7).
The Janus topology and the mesoporous structure were well retained after calcination at 700 °C under a N 2 atmosphere or prolonged sonification, indicating the high structural stability of the double-spherical Janus nanoparticles (Supplementary Figs. 8 and 9). Inorganic functional nanoparticles, such as UCNP@mSiO 2 &mPDA (UCNP, upconversion nanoparticle; Supplementary Fig. 10) and Fig. 11), can also be incorporated into the MSN compartments of the dual-mesoporous Janus structure, providing the Janus structures with functionalities such as light and magnetism. The mPDA section can also be replaced by other mesoporous polymers, such as mesoporous resorcinol formaldehyde resin ( Supplementary Fig. 12).

Formation mechanism of the mesoporous Janus nanoparticles
We next investigated the formation mechanism of the dual-mesoporous Janus structure. Although a series of factors affect the formation process, including pluronic block-copolymers (Supplementary Figs. 13 and 14), the water/ethanol ratio ( Supplementary Fig. 15), particle size and composition (Supplementary Figs. 16 and 17), TMB and CTAB were found to be the most crucial. Without the addition of TMB, a non-porous PDA shell is coated on the MSN nanoparticles, demonstrating that TMB serves as both the emulsion droplet for interfacial assembly of PDA and also as a directing agent for formation of the mesopore channels ( Supplementary Fig. 18). TMB is not the only oil phase that can serve this function, and the double-spherical nanoparticles can also be obtained by using other oil species, such as cyclohexane, triethyl benzene and toluene, indicating that it is the emulsion and not the specific oil that generates the Janus structure ( Supplementary  Fig. 19). The amount of cationic surfactant CTAB is found to play a crucial role in the anisotropic assembly of uniform mPDA hemispheres to form the Janus double-spherical nanoparticles. The MSNs are fabricated with a large amount of CTAB in the mesopores, and if they are used directly without extraction of CTAB, the mPDA fails to perform the anisotropic assembly, and a uniform MSN@mPDA core@shell structure is formed ( Supplementary Fig. 20). In the absence of CTAB, mPDA refuses to grow on the surface of the MSN nanospheres, leading to the formation of phase-separated mPDA and MSN nanoparticles ( Supplementary  Fig. 21a). When a suitable amount of CTAB (1.0 mg ml −1 ) is introduced, the Janus structure is achieved ( Supplementary Fig. 21b). When further increasing the CTAB concentration from 1.0 to 2.5 mg ml −1 , irregular MSN&mPDA structures with multiple mPDA islands are obtained (Supplementary Fig. 21c). The effect of CTAB on structure formation can be organized into a diagram ( Supplementary Fig. 22). Other cationic surfactants, including C 14 TAB, C 18 TAB and CTAC, can also work in the emulsion-oriented assembly process and lead to the formation of double-spherical MSN&mPDA nanoparticles. Anionic surfactants, on the other hand, fail to achieve similar results ( Supplementary Fig. 23).
The formation process for the dual-mesoporous Janus MSN&mPDA double-spherical nanoparticles was investigated (Fig. 3a,b). Ex situ cryo-TEM images clearly demonstrate the existence of oil droplets attached to the MSNs at the beginning of the reaction ( Supplementary  Fig. 24). Confocal laser scanning microscopy images also demonstrate a fluorescence signal from the MSN and TMB droplets (both are labelled with dyes; Supplementary Fig. 25). When the reaction has proceeded for 2 (hours) h, it can be observed that a low-contrast nanosphere has interacted with the solid MSN nanoparticle to form a Janus morphology (Fig. 3c). The nanosphere gradually turns into the mPDA hemisphere as the reaction proceeds (Fig. 3d), finally achieving the dual-mesoporous MSN&mPDA Janus nanostructure (Fig. 3e).
Based on the above results, we propose an emulsion-oriented assembly process for the formation of the dual-mesoporous MSN&mPDA Janus double-spherical nanoparticles (Fig. 3f). First, TMB is stabilized by CTAB and F-127 to form an emulsion-like droplet, then the TMB nano-droplets interact with the MSNs to form a double-spherical Janus structure. In this double-spherical structure, CTAB molecules function as the mediator, with their long hydrophobic chain stabilizing the TMB droplet, with the ammonium side against the hydrophilic MSN. The droplets then orient the F-127/PDA oligomer micelles to selectively assembly on the TMB. The mPDA hemispheres are grown and form in replacement of the TMB droplets, leading to the formation of peanut-like double-spherical Janus dual-mesoporous nanoparticles. This emulsion-oriented assembly process is supported by the observed experimental phenomena, as discussed in detail in the Supplementary Information (Supplementary Figs. [26][27][28][29].

Structure control of the mesoporous Janus nanoparticles
Precise manipulation of the pore size in both compartments of the Janus double-spherical nanoparticles can be achieved by varying the type of surfactant and oil phase 41,[44][45][46] . Uniform MSNs with pore sizes of ~3, 8, 15 and 25 nm (denoted MSN x , where x is the diameter of the pore in nanometres) were synthesized by altering the stirring speed and changing the oil phase from cyclohexane to octadecene and chlorobenzene ( Supplementary Fig. 30). The pore size of the mPDA hemispherical nanoparticles was also tuned in the range of ~5-50 nm (denoted mPDA y , where y is the diameter of the pore in nanometres) by changing the surfactant from pluronic F-127 to P-123 and F-108 ( Supplementary Fig. 31). In other words, the mesopore diameters of both the MSN and mPDA compartments of the Janus peanut-like nanoparticles can be tuned individually. As shown in Fig. 4, 16 types of mesoporous MSN x &mPDA y Janus double-spherical nanocomposite with controllable pore size can be synthesized. The size of the mesopores in the MSN domains is tuned to be ~3, 8, 15 or 25 nm, and the mesopores of the mPDA domains can be controlled to be 5, 15, 25 or 50 nm. TEM images of samples with different pore sizes in both compartments demonstrate the unique Janus nanostructure with tunable, dual, large mesopores (Fig. 4), which can be assembled into an extraordinary database of structures. Two groups of nitrogen sorption isotherm were investigated, the first comprising MSN 8 &mPDA y Janus nanoparticles with fixed mesopores in the MSN compartment and varied pore sizes in the mPDA compartment ( Supplementary Fig. 32), and the second being MSN x &mPDA 15 nano particles with varied pore sizes in the MSN compartment ( Supplementary Fig. 33). All the nitrogen sorption isotherms are typical type-IV curves, with a rapid increase of adsorption volume in  double intervals, evidencing the existence and controllability of the dual-mesoporous structure.

Selective loading of functional guests
As a result of the difference in the surface properties of silica and PDA and also the tunable mesopore sizes, selective loading of multiple guests in various compartments of the Janus mesoporous nanoparticles can be achieved. Typically, doxorubicin (DOX) is selectively loaded into the mesopore channels of fully spherical MSN compartments via pH-induced selectivity (Fig. 5a). In a neutral environment, DOX molecules are absorbed by both the MSN and mPDA. The DOX molecules load into the MSN compartments as a result of the capillary effect of the small-sized mesopore channels 26,47 , and their attraction towards the mPDA is mainly attributed to π-π stacking 48 . However, under weak acid conditions (pH 5), both the PDA and DOX molecules shift to a positive charge 48,49 , and the zeta potential of PDA shifts from −24 mV at neutral pH to 13 mV at pH 5. This charge repulsion prevents the DOX molecules from being absorbed by the mPDA (<10 mg g −1 ). Meanwhile, the isoelectric point of silica is at a pH of ~2, but at a pH of 5.5, the MSNs still possess a negative charge, so DOX can be loaded into the MSNs at a capacity of ~50 mg g −1 . Therefore, selective loading of DOX in the MSN compartment can be realized ( Supplementary  Fig. 34). Enzymes are also selectively loaded into the mPDA domains based on the difference in pore size between the two compartments (Fig. 5b). With Janus MSN 8 &mPDA 15 , the mesopore channels in the MSN compartment, with their diameter of ~8 nm, are smaller than the hydrodynamic sizes of enzymes (for example, glucose oxidase (GOx) has dimensions of 6.0 nm × 5.2 nm × 7.7 nm; ref. 50), and the loading of GOx in the MSN compartments is thus below the detection limit. Mesopores in the mPDA compartment have a sufficient size of 15 nm, and the GOx loading is ~30 wt%. However, with MSN 25 &mPDA 15 nanoparticles, with a silica mesopore diameter of 15 nm (which is larger than the enzymes used), the enzymes are loaded into the MSN compartment too (~16 wt%), and selective loading is not achievable. Thus, the different pore sizes on the two sides of the Janus architecture enable selective loading of enzymes into the mPDA compartment.

Biological logic gate establishment
Based on this selective loading of DOX and enzymes into the MSN and mPDA domains, respectively, biological logic gates were established based on the representative MSN 8 &mPDA 15 Janus mesoporous nanoparticles (Fig. 6a). Benzimidazole-functionalized MSNs with a pore size of ~8 nm were fabricated and used for the synthesis of Janus MSN&mPDA nanoparticles with a mesopore size of ~15 nm in the PDA compartment, then β-cyclodextrin-based supramolecular nanovalves were modified on the MSNs via benzimidazole-β-cyclodextrin supramolecular assembly. The benzimidazole-β-cyclodextrin supramolecular interaction is strong at neutral pH but breaks in acid conditions 37 . DOX molecules were selectively loaded into the MSN mesopore channels based on the pH-induced selective loading method and functioned as the 'output signal'. Enzymes with different catalytic properties were selectively loaded into the mPDA compartment based on the pore-size-induced selective loading method, enabling varied logic operations ( Supplementary  Fig. 35). The enzyme reacts with the substrates ('input signal'), and the products of this biological process are transmitted to the supramolecular nanovalves to determine whether or not to open the nanovalves. The MSN and mPDA are thus two processing units that together form an internal processor, which sequentially delivers the input signal through a series of biochemical reactions to the final output signal. As shown in Fig. 6b, by selecting different functional enzymes in the 'internal processor', different logic processes can be achieved. By varying the enzymes loaded into the mPDA compartment, three logic gates-YES, OR and AND-can be established using the large-mesoporous MSN&mPDA Janus nanoparticles as a general platform ( Supplementary Fig. 36). Controlled release of the output-signal DOX molecules in these three biological logic gates is carefully analysed in Supplementary Fig. 37. When loaded with GOx, the input signal (glucose) is catalysed by the GOx to form gluconic acid, lowering the pH and leading to functionalization of the signal converter (opening of the supramolecular nanovalves) and thus the output of signal (release of DOX). This response represents the basic YES logic. Logical responsive release of DOX from the dual-mesoporous MSN&mPDA Janus nanoparticles can be detected upon the addition of glucose, demonstrating successful establishment of the YES gate (Fig. 6c). It should be noted that the unique double-spherical mesoporous nanostructure also endows the established logic system with improved responsivity. The asymmetric double-spherical nanostructure has a substantially faster response rate than the mixture of MSN and mPDA nanoparticles. This effect is especially evident at a high glucose concentration of 10 mM, where a 50% higher output signal can be observed at an early release period of 15-30 min. We assume that the superior response rate can be attributed to the close contact of the two signal processors in the double-spherical nanostructure, which shortens the time required for signal transmission (Supplementary Fig. 38). Also, compared with the multi-island structured nanoparticles with multiple mPDA islands grown on one MSN, the delicate double-spherical nanoparticles with one mPDA hemisphere on one MSN have notably inhibited leakage of output signalling molecules of DOX (~40% less) when no input is provided. This leakage results in an increase in the sensitivity of the Janus nanoparticles ( Supplementary Fig. 39). The leakage is due to the multiple mPDA islands on the surface of MSN hindering the modification of supramolecular nanovalves on the MSNs, which are thus unable to effectively block signal molecules in the mesopores, leading to leakage of signal molecules, high background and low sensitivity. The other biological logic gates follow the same logical response, where only the correct input signals can trigger the internal processing reactions between materials loaded in the two compartments of the dual-mesoporous nanoparticles, resulting in the '1' output signal. When both GOx and esterase are loaded into the mPDA compartment, as well as glucose, esters such as ethyl butyrate also trigger the acid-forming reaction, leading to a '1' output. The release percentage of DOX with ethyl butyrate is ~50%, and that for glucose is ~30%, both successfully functioning as the '1' output signal. This is a typical OR logic (Fig. 6d).   When glucose dehydrogenase (GDH) is used, GDH responds only to the simultaneous presence of both glucose and nicotinamide adenine dinucleotide (NAD + ; Supplementary Fig. 40). Alone, the two factors cannot individually trigger a '1' output; the DOX release rates are only ~10% and ~15%, insufficient to reach the threshold value for a '1' output signal. However, when both glucose and NAD + are used as input signals, the DOX release rate can reach ~50%, enabling successfully operation as an AND logic gate (Fig. 6e). These results clearly indicate the successful establishment of various biological logic gates, demonstrating the dual-mesoporous MSN&mPDA Janus nanoparticles' facility as a substance for modular bio-computing system construction.
To verify the application potential of the biological logic gates, dual-mesoporous Janus MSN&mPDA nanoparticles with YES logic were used for glucose-responsive intracellular drug delivery. The dual-mesoporous Janus MSN&mPDA nanoparticles with selective loading of DOX and GOx (DOX/MSN&mPDA/GOx) were incubated with 4T1 cells. As can be seen from the confocal laser scanning microscopy images in Supplementary Fig. 41, without the glucose input, negligible DOX output was observed. In contrast, the representative red fluorescence from DOX was clearly observed after incubation in glucose-containing medium. These results clearly demonstrate the functionality of the biological logic gates in physiological environments, showing the feasibility of these nano-sized logic systems for use in new-generation smart nanomedicine.
Conventional logic gates mostly use enzymatic catalysis as one of the information processors, because the non-porous or single-porous (only one set of pore channels) structure cannot realize the independent storage and sequential release of multiple intermediate and output signalling molecules (Supplementary Fig. 42). To further highlight the advantages of this dual-mesoporous Janus structure, we constructed a single-particle biological logic gate based on the tandem release of multiple intermediate and output signalling molecules ( Supplementary  Fig. 43). We selectively loaded TCEP (tris(2-carboxyethyl)phosphine) and a modified benzimidazole-cyclodextrin gatekeeper in the MSN compartment. Long-chain polymeric gatekeepers containing disulfide bonds (O-(2-mercaptoethyl)-O′-methyl-hexa(ethylene glycol)) were selectively modified on the mPDA compartment to block the safranin O dyes in the mesopore channels. In this logic-gate system, with an input signal of acid (H + ), the benzimidazole-cyclodextrin valves on the MSN side were opened to release TCEP intermediate signalling molecules. The TCEP from the MSNs sequentially broke the disulfide bonds in the polymer valves on the mPDA unit, triggering the release of a safranin O output signal ( Supplementary Fig. 44). Compared with signal transfer based on enzyme-catalysed reactions, tandem sequential release of multiple signalling molecules is much closer to the real situation of signalling carried out in organisms. It can also greatly expand the scope of logic systems based on diverse kinds of gatekeepers 51,52 .

Conclusion
Mesoporous Janus MSN&mPDA double-spherical nanoparticles with dual large and tunable mesopores have successfully been synthesized via an emulsion-oriented assembly method. In this approach, oil droplets first interact with MSNs to form double-spherical structures and then orient the selective encapsulation of mPDA to form Janus double-spherical MSN&mPDA nanoparticles. The emulsion-oriented assembly strategy is quite different from the previously reported anisotropic growth strategy [19][20][21] , which is suitable for the synthesis of highly ordered mesoporous structures, such as cubic (Pm-3n), two-dimensional hexagonal (p6m) mesostructures. The component of the asymmetric domains obtained with the anisotropic growth strategy is mainly silica or organosilica. However, in this work, the mesostructure of the asymmetric mPDA domains is disordered, and the component is polymer. So, the formation process and mechanism are quite different, with the TMB oil phase playing a crucial role in the formation of the asymmetric structure.
The fabricated dual-mesoporous Janus nanoparticles include a monodispersed MSN nanosphere (~150 nm in diameter) with tunable mesopores from ~3 to ~25 nm and an mPDA hemisphere with a uniform diameter of ~120 nm and alterable pore size from ~5 to ~50 nm. We have demonstrated the individual and fine manipulation of pore size on both sides of the dual-mesoporous Janus double-spherical nanomaterials. Based on the unique asymmetric double-spherical morphology and dual large tunable mesopores, biological logic gates with YES, OR and AND logic have been established successfully, demonstrating the ability of the Janus nanoparticles to form the basis for modular bio-computing-system construction. The Janus structure's unique architecture enhances the sensitivity and responsivity for establishing logic systems. A logic system with consecutive valve-opening and matter-releasing reactions within one single nanoparticle has been established based on the dual-mesoporous structure. This will greatly expand the scope of the design and complexity of biological logic gates. Such Janus MSN&mPDA nanoparticles with dual large and tunable mesopores and selective functionalization are highly extendable to other applications and have a great potential to become a multifunctional platform not only for biological logic gates but also for catalysis, energy storage, sensing and environmental remediation.

Online content
Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41557-023-01183-4.

Fabrication of MSNs
MSNs were synthesized using a bi-phase method according to previous reports, with brief modifications. In a typical synthesis of MSNs with a pore size of ~8 nm, 3.00 g of CTAB, 100 μl of triethanolamine and 60.0 ml of H 2 O were added to a 100-ml three-necked flask. The mixture was stirred until clear, then transferred into an oil bath at 60 °C. A Teflon rotor (length, 2.0 cm) was used, and the stirring rate was set to 150 r.p.m. A mixture of 4.0 ml of tetraethyl orthosilicate and 16.0 ml of cyclohexane was added carefully on top of the solution to form a bi-phase. The reaction was allowed to proceed for 48 h, then centrifuged and washed with water and ethanol. The synthesis of MSNs with other pore sizes is described in the Supplementary Information.

Template CTAB extraction from MSNs
MSNs were dispersed in a 5.0 mg ml −1 NH 4 NO 3 ethanol solution and refluxed at 60 °C for 4 h. This process was repeated three times to guarantee complete extraction of CTAB.

Fabrication of MSN&mPDA Janus nanoparticles
The MSN&mPDA Janus nanoparticles were fabricated using an emulsion-oriented assembly process. Typically, 5.0 mg of MSNs with CTAB extracted were dispersed in 5.0 ml of H 2 O and 5.0 ml of ethanol, then 10.0 mg of CTAB, 200 mg of F-127 and 100 mg of dopamine were added, then stirred to form a clear solution. TMB (1.0 ml) was added, then the mixture was stirred for 30 min to form a milky white emulsion, before adding 10.0 mg of tris(hydroxymethyl)aminomethane. The reaction was left to proceed overnight, then centrifuged and washed with water and ethanol. The synthesis of MSN&mPDA nanoparticles with other pore sizes is described in the Supplementary Information.

Biological logic gate establishment
To establish the biological logic gate, MSN&mPDA nanoparticles functionalized with benzimidazole moieties in the MSN section were first fabricated. In a typical synthesis, 20.0 mg of MSNs, with CTAB extracted, was dispersed in 10.0 ml of acetonitrile, then 100 μl of (3-iodopropyl)trimethoxysilane was added. The mixture was stirred overnight, then centrifuged and washed with acetonitrile to obtain the iodine-propyl-functionalized MSNs. The MSNs functionalized with iodine propyl were then dispersed in 8.0 ml of benzimidazole toluene (1.0 mg ml −1 ) solution at 80 °C, then 24.0 ml of trimethylamine was added, and the suspension was stirred for 72 h to achieve the benzimidazole-moiety-functionalized MSNs 3 . The emulsion-oriented assembly of mPDA was the same as above, and MSN&mPDA nanoparticles functionalized with benzimidazole moieties in the MSN section were obtained.
To achieve selective DOX loading, hydrochloric acid was added to tune the pH of 4.0 mg ml −1 DOX solution to ~5.5. The Janus MSN&mPDA nanoparticles were then added at a concentration of 2.0 mg ml −1 . The solution was stirred in the dark for 24 h, then centrifuged to achieve MSN&mPDA loaded with DOX only in the MSN section (denoted as DOX/MSN&mPDA). To 'cap' the silica mesopores, 10.0 mg of DOX/ MSN&mPDA nanoparticles were then suspended in 10.0 ml of H 2 O, then 5.0 mg of β-cyclodextrin was added. After stirring overnight, the nanoparticles were centrifuged and washed with water. DOX loading amounts were determined by examining the UV-vis absorption of the supernatant at 450 nm after centrifuging the nanoparticles. The loading amounts of DOX in the MSNs and mPDA were evaluated by using MSNs and mPDA as nanocarriers, respectively.
To achieve mPDA selective modification of enzymes, the DOX/MSN&mPDA nanoparticles were dispersed in 10.0 ml of H 2 O, and 10.0 mg of GOx was added. After stirring for 24 h, the nanoparticles were centrifuged and washed with water to obtain the Janus MSN&mPDA nanoparticles with DOX loaded in the MSN section and enzyme-modified in the mPDA section (denoted DOX/MSN&mPDA/ GOx). The loading of other types of enzyme for different biological logic gates was processed in the same manner.

Data availability
Data supporting the findings of this study are available within the Article and the associated Supplementary Information. Source data are provided with this paper.