The simultaneous evolution of multiple aptamers can drastically increase the speed of aptamer discovery. Most previous studies used the same concentration for different targets, leading to the dominance of the libraries by one or a few aptamers and a low success rate. To foster the best aptamers to grow independently in the sequence space, it is important to (1) use low target concentrations close to their dissociation constants and (2) stop at an early round before any sequence starts to dominate. In this study, we demonstrate this affinity-guided selection concept using the capture-SELEX method to isolate aptamers for four important purines: guanine (5 μM), xanthine (50 μM), hypoxanthine (10 μM), and adenine (10 μM). The round 9 library was split, and in round 10, the four targets were individually used to elute the binding sequences. Using thioflavin T fluorescence spectroscopy and isothermal titration calorimetry, we confirmed highly selective aptamers for xanthine, guanine, and adenine. These aptamers have Kd values below 1 μM and around 100-fold selectivity against most competing analytes, and they compare favorably with existing RNA aptamers and riboswitches. A separate selection was performed using hypoxanthine alone, and no selective aptamer was achieved, even with negative selection, explaining the lack of its aptamer in our mixed selection. This affinity-guided multiplex SELEX study offers fundamental insights into aptamer selection and provides high-quality aptamers for three important purines.

Abstract Salicylic acid (SA) is a hydrolysis product and an active form of aspirin, and SA is found in a range of fruits and other food products. For food and drug and analysis there is a strong desire to detect SA. Since SA is a very small molecule, aptamers have advantages over antibodies for its detection. In this work, we used the library‐immobilization capture‐SELEX method to isolate aptamers for SA. After 17 rounds of selection, two main families of aptamers were isolated. The SA1 aptamer from family 1 has a Kd of 5.8 μM from a thioflavin T (ThT) fluorescence assay and 26.7 μM from isothermal titration calorimetry. The binding of other sequences was weaker compared to SA1. Based on mutation studies, the two conserved regions of SA1 were connected by two stems. Using ThT as a stain, a label‐free fluorescent sensor was tested for the detection of SA with a detection limit of 2.2 μM. A few similar molecules were tested including aspirin, and only p‐hydroxybenzoic acid showed a weak binding, indicating the high specificity of the SA1 aptamer. Finally, the SA1 aptamer was also tested in tomato juice and a similar binding performance was achieved.

Using DNA staining dyes such as SYBR Green I (SGI) and thioflavin T (ThT) to perform label-free detection of aptamer binding has been performed for a long time for both binding assays and biosensor development. Since these dyes are cationic, they can also adsorb to the wall of reaction vessels leading to unstable signals and even false interpretations of the results. In this work, the stability of the signal was first evaluated using ThT and the classic adenosine aptamer. In a polystyrene microplate, a drop in fluorescence was observed even when non-binding targets or water were added, whereas a more stable signal was achieved in a quartz cuvette. Equilibrating the system can also improve signal stability. In addition, a few polymers and surfactants were also screened, and 0.01% Triton X-100 was found to have the best protection effect against fluorescence signal decrease due to dye adsorption. Three aptamers for Hg2+, adenosine, and cortisol were tested for their sensitivity and signal stability in the absence and presence of Triton X-100. In each case, the sensitivity was similar, whereas the signal stability was better for the surfactant. This study indicates that careful control experiments need to be designed to ensure reliable results and that the reliability can be improved by using Triton X-100 and a long equilibration time.

Tetracyclines are a group of very important antibiotics that are still in use. To extract, detect, and remove tetracyclines from the environment, various nanomaterials have been employed. Although gold nanoparticles (AuNPs) are a commonly cited material for these purposes, a fundamental understanding of these tetracycline-AuNP systems is still limited. In this work, the adsorption of tetracycline, oxytetracycline, and doxycycline to AuNPs was studied. The effect on the colloidal stability of AuNPs, adsorption kinetics, and the resulting adsorption isotherms was measured. While millimolar concentrations of the tetracyclines can cause aggregation of AuNPs, saturated monolayer adsorption was achieved with low micromolar concen-trations of the tetracyclines. Adsorption was instantaneous, and adsorption to AuNPs enhanced their intrinsic fluorescence instead of quenching. With the assumption that aptamer/target complexes cannot be easily adsorbed by AuNPs compared to free aptamers, a label-free colorimetric detection method was tested. While the label-free sensor showed target-dependent aggregation of AuNPs, a nonbinding mutant aptamer showed the same trend, suggesting that the color change did not re-flect aptamer adsorption but other events such as target adsorption. This study indicates the importance of the fundamental understanding of target/AuNP interactions to correctly design aptamer and AuNP-based label-free biosensors.

To improve tumor destruction and minimize adverse effects to healthy tissues, image-guided radiation therapy (IGRT) has been developed to allow for the accurate delivery of radiation energy to tumor sites facilitated by real-time imaging. Nevertheless, the current IGRT platform still suffers from the limitation of poor tissue contrast, resulting in the incidental irradiation of healthy tissue. Gold nanoparticles (GNPs) have been identified as promising candidates to simultaneously improve both radiotherapy and imaging, thereby improving both the accuracy and safety of IGRT. However, despite much preclinical study, little clinical progress has been made due to uncertainty over GNP toxicity. Herein, we demonstrate the great potential of using GNP-coated liposomes, i.e., Lipogold, which combine the advantages of both large and small nanoparticles into one multifunctional formulation, as an ideal platform for IGRT. When irradiated with low doses (<2 Gy) of therapeutic X-rays, Lipogold induced a significant radiosensitization effect for PC-3 prostate cancer cells, which are moderately radiation-resistant. When imaged with computed tomography (CT), Lipogold was also found to possess consistent X-ray contrast of similar to 18-23 HU/mg across tube X-ray voltages (70-140 kVp), which could be boosted via the encapsulation of a small-molecule contrast agent containing iodine.

Recently, many nanomaterials such as Fe3O4, CeO2, and gold nanoparticles have been reported to have enzyme-like activities and they are called nanozymes. Although these nanozymes have oxidase or peroxidase-like activities, they can catalyze the oxidation of many substrates and thus lack the specificity expected for enzymes. The selectivity of nanozymes can be significantly enhanced up to 100-fold by coating them with a molecularly imprinted polymer (MIP) layer. Since MIP creates specific binding pockets for the imprinted substrate, the imprinted molecules can be enriched, selectively access the catalytic core, and be oxidized, while other substrates are blocked from accessing the nanozyme surface. In this chapter, the detailed protocol for the preparation of the MIP-coated Fe3O4 peroxidase-mimicking nanozymes is described. In addition, some procedures needing special attention are described in detail, which will facilitate the applications of MIP-coated nanozymes in analytical, biomedical, and environmental fields.

Nanozymes are nanomaterials mimicking the activity of natural enzymes, while most nanozymes lack substrate specificity. Molecular imprinting on nanozymes provides a simple solution to this problem, and the catalytic activity is also enhanced. To understand enhanced activity, a surface science approach is taken by dissecting the nanozyme reaction into adsorption of substrates, reaction, and product release. Each step is individually studied using reaction kinetics measurement, dynamic light scattering, UV-vis spectrometry. Enrichment of local substrate concentration due to imprinting is around 8-fold, and increased substrate concentration could contribute to increased activity. Diffusion of the substrate across the imprinted gel layer is studied by a pre-incubation experiment, also highlighting the difference between imprinted and non-imprinted gel layers. The activation energy is measured and a substrate-imprinted sample had the lowest activation energy of 13.8 kJ mol-1. Product release is also improved after imprinting as indicated by isothermal titration calorimetry using samples respectively imprinted with the substrate and the product. This study has rationalized improved activity and specificity of molecularly imprinted nanozymes and may guide further rational design of such materials.

A hallmark of nanoscience is size-dependent and distance-dependent physical properties. Although most previous studies focused on optical properties, which are often tuned at nanometer scale, we herein report on the interaction between halide-capped gold nanoparticles (AuNPs) and phosphocholine (PC) liposomes at the sub-Angstrom level. Halide-capped AuNPs are adsorbed by PC liposomes attributable to van der Waals force. Iodide-capped AuNPs interact much more weakly with the liposomes compared with bromide- and chloride-capped AuNPs, as indicated by a liposome leakage assay and differential scanning calorimetry. This is explained by the slightly larger size of iodide separating the AuNP core more from the liposome surface. Cryo-transmission electron microscopy indicates that the liposomes remain intact when mixed with these halide-capped AuNPs of 13 or 70 nm in diameter. Other even larger ligands, including small thiol compounds, DNA oligonucleotides, proteins, and polymers, fully blocked the interaction, whereas AuNPs dispersed in noninteracting ions, including fluoride, phosphate, perchlorate, nitrate, sulfate, and bicarbonate, are still adsorbed strongly by 1,2-dioleoyl- sn-glycero-3-phosphocholine liposomes. Taken together, halides can be used to control interparticle distances at an extremely small scale with remarkable effects on materials properties, allowing surface probing, biosensor development, and fundamental surface science studies.

TiO2 and SiO2 are very useful materials for building biointerfaces. A particularly interesting aspect is their interaction with lipid bilayers. Many past research efforts focused on phosphocholine (PC) lipids, which form supported lipid bilayers (SLB) on SiO2 at physiological conditions but are adsorbed as intact liposomes on TiO2. Low pH was required to form PC SLBs on TiO2. This work intends to understand the surface forces and chemistry responsible for such differences. Two charge neutral lipids: 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe) and two negatively charged lipids: 1,2-dioleoyl- sn-glycero-3-phospho-l-serine (DOPS) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP) were used. Using calcein leakage assays, adsorption measurement, cryo-TEM, and washing, we concluded that charge is the dominating factor on SiO2. The two neutral lipids form SLB on SiO2 at pH 3 and 7, but the two negatively charged ones cannot form. On TiO2, both charge and coordination chemistry are important. The two anionic lipids formed SLB from pH 3 to 10. DOCP had stronger affinity than DOPS likely due to the tighter terminal phosphate binding of the former. The two neutral liposomes formed SLB only at pH 3, where phosphate interaction and van der Waals force are deemed important. The pH 3 prepared TiO2 DOPC SLBs are destabilized at neutral pH, indicating the reversible nature of the interaction. This work has provided new insights into two important materials interacting with common liposomes, which are important for reproducible biosensing, device fabrication, and drug delivery applications.

Perfluorinated compounds (PFCs) are emerging persistent environmental contaminants that may be toxic to animals and humans. To gain fundamental insights into the mechanism of their toxicity, the interactions of phosphocholine (PC) liposomes as model membranes were studied with three types of PFCs, including perfluorooctanoic acid, perfluorooctane sulfonate, and perfluorohexanesulfonic acid potassium salt, together with three common surfactants: sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and sodium 1-heptanesulfonate (SHS). The interactions were systematically characterized by zeta potential measurement, dynamic light scattering, negative-stain transmission electron microscopy, and fluorescence spectroscopy. Unmodified liposomes, calcein-loaded liposomes, and Laurdan dye-embedded liposomes were all tested. By gradually increasing the temperature, the three PFCs and SHS decreased the leakage of calcein-loaded 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes, whereas SDS and CTAB increased the leakage. The PFCs that affected the lipid membranes stronger than SHS were attributable to their perfluoroalkyl carbon chains. Packing of the lipids was further studied using Laurdan dye as a probe. Calcein leakage tests also indicated that PFCs inhibited lipid membrane leakage induced by inorganic nanoparticles such as silica and gold nanoparticles. This study confirmed the similar effect of the PFCs as cholesterol in affecting membrane properties and would be helpful for understanding the interaction mechanism of PFCs and cell membranes.

Polymersomes are vesicles formed by self-assembled amphiphilic block copolymers. Polymersomes generally have better stability than liposomes and they have been widely used in making drug delivery vehicles. In this work, the interaction between two types of polymersomes and citrate-capped gold nanoparticles (AuNPs) was studied. The following two polymers: poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline) (called P1) and poly(butadiene-b-ethylene oxide) (called P2) were respectively used to form polymersomes. While P1 only formed spherical vesicle structures, worm-like structures were also observed with P2 as indicated by cryo-TEM. Both polymersomes adsorbed AuNPs leading to their subsequent aggregation. A lower polymersome concentration produced more obvious aggregation of AuNPs as judged from the color change. Capping AuNPs with glutathione inhibited adsorption of AuNPs. Considering the surface property of the polymers, the interaction with AuNPs was likely due to van der Waals forces. P1 polymersomes encapsulated calcein stably and AuNPs did not induce leakage. The P1/AuNP complex was more efficiently internalized by HeLa cells compared to free P1 polymersomes, further indicating a stable adsorption under cell culture conditions. In summary, this work indicates citrate-capped AuNPs form stable adsorption complexes with these polymersomes and their interactions have been explored.

We herein report a three-component cell-mimicking structure with a peroxidase-like iron oxide nanozyme as the nucleus, a molecularly imprinted hydrogel shell as cytoplasm, and a lipid bilayer membrane. The structure was characterized by cryo and negative stain TEM and also by a calcein leakage test. By introducing charged monomers, the gel shell can swell or shrink in response to salt concentration. By lowering the salt concentration, the gradual "analog" gel volume change was reflected in a switch-like "digital" colorimetric output by the burst of membrane and oxidation of substrates such as 3,3',5,5'-tetramethylbenzidine (TMB). Controlled access was also achieved by using melittin to insert channels cross the membrane, and selective molecular transport was realized by the molecularly imprinted gel. The functions of each component are coupled, and this sophisticated tripartite structure provides a new platform for modular design of new materials. Our cell-mimicking structure is functional and it is complementary to the current protocell work that aims to understand the origin of life.

Zwitterionic phosphocholine (PC) liposomes are widely used in drug delivery because of their high biocompatibility and long blood circulation time. We herein report that by flipping the direction of the PC dipole, the resulting choline phosphate (CPe) liposomes have an even longer circulation time, as confirmed at both cellular and animal model levels. Even when 33% cholesterol was included in the lipid formulation with a poly(ethylene glycol) layer, the CPe liposome still had a longer blood circulation time. Isothermal titration calorimetry indicates a lack of protein adsorption or PC membrane attachment for the CPe liposomes. This is different from the previously reported adhesion of CP polymers to PC lipid membranes, which may be attributed to the different ways of displaying the CP headgroup. With a longer blood circulation time, the CPe liposomes accumulated in tumors more easily than PC liposomes, which is likely due to the enhanced permeation and retention effect and tumor cell uptake. This study provides key insights into zwitterionic biointerfaces for biomedical, analytical, and materials applications.

Enzyme-mimicking nanomaterials (nanozymes) are more cost-effective and robust than protein enzymes, but they lack specificity. Herein, molecularly imprinted polymers were grown on Fe3O4 nanozymes with peroxidase-like activity to create substrate binding pockets. Electron microscopy confirmed a shell of nanogel. By imprinting with an adsorbed substrate, moderate specificity was achieved with neutral monomers. Further introducing charged monomers led to nearly 100-fold specificity for the imprinted substrate over the nonimprinted compared to that of bare Fe3O4. Selective substrate binding was further confirmed by isothermal titration calorimetry. The same method was also successfully applied for imprinting on gold nanoparticles (peroxidase mimics) and nanoceria (oxidase mimics). Molecular imprinting furthers the functional enzyme mimicking aspect of nanozymes, and such hybrid materials will find applications in biosensor development, separation, environmental remediation, and drug delivery.

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo-TEM, and ζ-potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3 O4 , TiO2 , ZrO2 , Y2 O3 , ITO, In2 O3 , and Mn2 O3 ) form supported bilayers only with DOCP. Type 2 (SiO2 ) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3 O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.