High-Density Au Anchored to Ti3C2-Based Colorimetric-Fluorescence Dual-Mode Lateral Flow Immunoassay for All-Domain-Enhanced Performance and Signal Intercalibration.

In this work, a novel MXene-Au nanoparticle (Ti3C2@Au) was synthesized with a high molar extinction coefficient, strong fluorescence quenching ability, ultrahigh antibody affinity, high stability, and good dispersibility, and it was used to develop a colorimetric-fluorescence dual-mode lateral flow immunoassay (LFIA). The detection limits of this method for the detection of dexamethasone in milk, beef, and pork were 0.0018, 0.12, and 0.084 µg/kg in the "turn-off" mode (colorimetric signal), and 0.0013, 0.080, and 0.070 µg/kg in the "turn-on" mode (fluorescent signal), respectively, which was up to 231-fold more sensitive compared with that of the reported LFIAs. The recovery rates ranged from 81.1-113.7%, and 89.2-115.4%, with the coefficients of variation ranging from 1.4-15.0%, and 1.9-14.8%, respectively. The results of the LC-MS/MS confirmation test on 30 real samples had a good correlation with that of our established method (R2 > 0.97). This work not only developed novel nanocarriers for antibody-based LFIA but also ensured high-performance detection.

Kinetic ITC of DNA Aptamers Binding for Small Molecules and Implications for Binding Assays and Biosensors.

The determination of kon and koff values through kinetic analysis is crucial for understanding the intricacies of aptamer-target binding interactions. By employing kinetic ITC, we systematically analyzed a range of ITC data of various aptamers. Upon plotting their kon and koff values as a function of their Kd values, a notable trend emerged. Across a range of Kd values spanning from 28 nM to 864 µM, the kon value decreased from 2×105 M-1 s-1 to 96 M-1 s-1, whereas the koff value increased from 1.03×10-3 s-1 to 0.012 s-1. Thus, both kon and koff contributed to the change of Kd in the same direction, although the range of kon change was larger. Since experiments are often run at close to the Kd value, this concentration effect also played an important role in the observed binding kinetics. The effect of these kinetic parameters on two common sensing mechanisms, including aptamer beacons and strand-displacement assays, are discussed. This work has provided the kinetic values of small molecule binding aptamers and offered insights into aptamer-based biosensors.

Exploring the Lower Limit of Target Concentration in Capture-SELEX Using Guanine as a Model Target.

During an aptamer selection, using a lower target concentration may result in aptamers with a higher binding affinity. Consequently, this begs the question of whether there is a lower limit for target concentration. In this work, we conducted three aptamer selections using 5 µM, 500 nM and 50 nM guanine as the targets, respectively. Successful enrichment of the same guanine aptamers was achieved at both 5 µM and 500 nM guanine, but not with 50 nM. Using 5 µM guanine, the aptamer was enriched in eight rounds of selection, compared to that for 500 nM, which was accomplished in 17 rounds. We discuss the relation of optimal target concentration to the observed Kd value of the resulting aptamers, of which the highest affinity aptamer had a measured Kd of 200 nM. Additionally, we investigated the binding of the aptamers through mutation studies, revealing a critical cytosine. Mutating this cytosine to a thymine switched the selectivity from guanine to adenine, which is reminiscent of the guanine riboswitch. This study revealed a limit in using low target concentration, and the insights described in this article will be useful for guiding the choice of target concentration during capture-SELEX.

Selection and Characterization of DNA Aptamers for Cytidine and Uridine.

Cytidine and uridine are two essential pyrimidine ribonucleotides, and accurate detection of these nucleosides holds significant biological importance. While many aptamers were reported to bind purines, little success was achieved for pyrimidine binding. This study employs the library-immobilization capture-SELEX technique to isolate aptamers capable of selectively binding to cytidine and uridine. First, a selection was performed using a mixture of cytidine and uridine as the target. This selection led to the isolation of a highly selective aptamer for cytidine with a dissociation constant (Kd ) of 0.9 µM as determined by isothermal titration calorimetry (ITC). In addition, a dual-recognition aptamer was also discovered, which exhibited selective binding to both cytidine and uridine. Subsequently, a separate selection was carried out using uridine as the sole target, and the resulting uridine aptamer displayed a Kd of 4 µM based on a thioflavin T fluorescence assay and a Kd of 102 µM based on ITC. These aptamers do not have a strict requirement of metal ions for binding, and they showed excellent selectivity since no binding was observed with their nucleobases or nucleotides. This study has resulted three aptamers for pyrimidines, which can be employed in biosensors and DNA switches.

An Ultrahigh Affinity DNA Aptamer for Quinine and Its Intrinsic Fluorescence Based Label-free Detection.

Measuring quinine is critical for the detection of its overdose, understanding its pharmacological and toxicological effects, and monitoring its pollution. While a previously reported aptamer named MN4 can bind quinine, it was not selected for it, leading to compromised binding affinity and specificity. In this work, a new quinine aptamer was isolated using the library immobilization capture-SELEX technique. The Q1 aptamer has a Kd value of 10 nM determined by an isothermal titration calorimetry experiment and 45 nM in a fluorescence binding assay. A 3.5 nM quinine limit of detection was obtained based on the aptamer binding-induced quenching of the intrinsic fluorescence of quinine. A large blue shift in fluorescence was observed for quinine upon binding to Q1, whereas binding to MN4 led to a very small red shift, indicating different ways of quinine binding by these two aptamers. Q1 did not bind cocaine based on NMR spectroscopy and fluorescence assays also indicated excellent selectivity against other tested molecules. This work has supplied a high affinity aptamer for quinine that can be useful for its detection and fundamental aptamer binding studies. It also highlights the advantages of using capture-SELEX to isolate aptamers for small molecules.

Gold Nanoparticles in Serum and Milk and Their Effect for Label-Free Colorimetric Sensing of DNA.

Label-free gold nanoparticle (AuNP)-based colorimetric biosensors have been widely used for the detection of DNA. However, the effect of the biological sample matrix has not been fully explored. In this work, we investigated the salt-induced aggregation of AuNPs as well as DNA adsorption in serum and milk. AuNPs of 13, 30, and 50 nm were used as probes. The detection was successful only in a clean buffer but failed in serum or milk. Serum and milk exhibited excellent protective properties, even 250 mM NaCl added did not induce the aggregation of AuNPs. After centrifugation of milk, the supernatant did not protect the AuNPs, whereas the redispersed pellet showed protection. The limit concentration of serum to prevent AuNPs from aggregating was 0.04% for 13 nm AuNPs and 0.01% serum for 50 nm AuNPs. In addition, serum reduced DNA adsorption, and the DNA was adsorbed to the protein corona instead of directly to the AuNP surface. These two factors can explain the difficulty of detection in protein-containing samples. This study articulates the adsorption of proteins by AuNPs in biological samples and offers useful insights into the biosensor design.

Adsorption of DNA and Aptamers to Sodium Urate Crystals and Inhibition of Crystal Growth.

An elevated level of blood uric acid (UA) can cause the formation of kidney stones, gout, and other diseases. We recently isolated a few DNA aptamers that can selectively bind to UA. In this work, we investigated the adsorption of a UA aptamer and random sequence DNA onto sodium urate crystals. Both DNA strands adsorbed similarly to urate crystals. In addition, both the UA aptamer and random DNA can inhibit the growth of urate crystals, suggesting a nonspecific adsorption mechanism rather than specific aptamer binding. In the presence of 500 nM DNA, the growth of needle-like sodium urate crystals was inhibited, and the crystals appeared granular after 6 h. To understand the mechanism of DNA adsorption, a few chemicals were added to desorb DNA. DNA bases contributed more to the adsorption than the phosphate backbone. Surfactants induced significant DNA desorption. Finally, DNA could also be adsorbed onto real UA kidney stones. This study provides essential insights into the interactions between DNA oligonucleotides and urate crystals, including the inhibition of growth and interface effects of DNA on sodium urate crystals.

Intramolecular aptamer switches.

Aptamer switches as effective biosensing tools have become a focal point of research in engineered aptasensors. Intramolecular aptamer switches are more versatile, affordable, and simpler than classical "open-close" and strand displacement-based aptamer switches. Recently, many new aptamers with an overall hairpin structure have been reported. In this study, intramolecular aptamer switches were developed by adding new base pairs to the end of aptamers. The additional nucleotides can pair with the internal domains of the aptamer, causing a change in its conformation from the original secondary structure without a target. When a target binds to an aptamer, a marked change in the structure of the aptamer is expected. As models for testing this intramolecular aptamer switch idea, aptamers of oxytetracycline (OTC), 17ß-estradiol (E2), and adenosine were employed. When the additional base pairs are too long, binding the target to the aptamer becomes more challenging. This research offers valuable insights into the development of intramolecular aptamer switches and their potential applications in biosensor design.

Characterization of nanozyme kinetics for highly sensitive detection.

Nanozymes have been widely used as enzyme substitutes. Based on a comprehensive literature survey of 261 publications, we report the significant differences in the Michaelis-Menten constants (Km) between peroxidase-mimicking nanozymes and horseradish peroxidase (HRP). Further, these differences were not considered in more than 60% of the publications for analytical developments. As a result, nanozymes' catalytic activity is limited, resulting in a potentially higher limit of detection (LOD). We used a peroxidase-mimicking Au@Pt nanozyme, which has Km for TMB comparable with HRP and three orders of magnitude higher Km for H2O2. Using the Au@Pt nanozyme as a label for immunoassays, non-optimized nanozyme substrate concentrations led to 30 times higher LOD compared to optimized conditions. The results confirm the necessity of measuring nanozymes' kinetic parameters and the corresponding adjustment of substrate concentrations for highly sensitive detection.

Selection of DNA aptamers for detecting metronidazole and ibuprofen: two common additives in soft drinks.

To enhance the effects of some functional soft drinks, drugs, especially metronidazole (MNZ) and ibuprofen (IBF), are often illegally added. This poses a serious threat to the health of consumers. Therefore, developing simple and rapid detection methods for these additives is crucial. In this study, DNA aptamers of metronidazole and ibuprofen were selected using the library-immobilized method. The best aptamer for metronidazole, named MNZ-1, has a dissociation constant (Kd) value of 4.9 µM and the aptamer for ibuprofen, named IBF-1, shows a Kd of 9.3 µM, as determined by the thioflavin T (ThT) fluorescence assay. The Kd values measured using isothermal titration calorimetry (ITC) were 17.0 µM and 66.7 µM for these two aptamers, respectively. Selectivity experiments indicate that MNZ-1 demonstrates very weak binding to structurally similar drugs, whereas IBF-1 exhibits binding capability to some structurally similar compounds comparable to ibuprofen, enabling the simultaneous detection of these types of drugs. Neither MNZ-1 nor IBF-1 binds to other common drugs. Using ThT, a label-free fluorescent detection method was developed for metronidazole and ibuprofen in soft drinks, showing limits of detection (LODs) of 0.6 µM and 4.7 µM, respectively. Owing to their small size and well-defined secondary structures, these aptamers are expected to be utilized in analytical applications for food and environmental monitoring.

DNA aptamers for common buffer molecules: possibility of buffer interference in SELEX.

During a typical aptamer selection experiment, buffer molecules are used at the 10 to 50 mM range, whereas target molecules could be used at much lower concentrations even in low µM levels. Therefore, doubts existed regarding the potential enrichment of buffer binding aptamers, particularly for failed selections that cannot validate binding of enriched sequences. In this study, we used two common buffer molecules, Tris and HEPES, as target molecules. While we successfully isolated aptamers for Tris buffer, our attempts to generate aptamers for HEPES buffer failed. Thioflavin T (ThT) fluorescence spectroscopy showed the dissociation constant (Kd) of the Tris buffer aptamer to be 2.9 mM, while isothermal titration calorimetry showed a Kd of 43 µM. NMR spectroscopy also confirmed aptamer binding. Finally, we discussed the implications of this buffer selection work and recommended the use of certain buffers.

Rapid Thermal Drying Synthesis of Nonthiolated Spherical Nucleic Acids with Stability Rivaling Thiolated DNA.

Attaching DNA oligonucleotides to gold nanoparticles (AuNPs) to prepare spherical nucleic acids (SNAs) has offered tremendous insights into surface chemistry with resulting bioconjugates serving as critical reagents in biosensors and nanotechnology. While thiolated DNA is generally required to achieve stable conjugates, we herein communicate that using a thermal drying method, a high DNA density and excellent SNA stability was achieved using nonthiolated DNA, rivaling the performance of thiolated DNA such as surviving 1 M NaCl, 2 month stability in 0.3 M NaCl and working in 50 % serum. A poly-adenine block with as few as two consecutive terminal adenine bases is sufficient for anchoring on AuNPs. By side-by-side comparison with the salt-aging method, the conjugation mechanism was attributed to competitive adenine adsorption at high temperature along with an extremely high DNA concentration upon drying. Bioanalytical applications of nonthiolated SNAs were validated in both solution and paper-based sensor platforms, facilitating cost-effective applications for SNAs.

Selective Hemin Binding by a Non-G-quadruplex Aptamer with Higher Affinity and Better Peroxidase-like Activity.

Previous aptamers for porphyrins and metalloporphyrins were all guanine-rich sequences that can fold in G-quadruplex structures. Due to stacking-based binding, these aptamers can hardly tell different porphyrins apart, and they can also bind other planar molecules, hindering their practical applications. In this work, we used the capture selection method to obtain aptamers for hemin and protoporphyrin IX (PPIX). The hemin aptamer (Hem1) features two highly conserved repeating binding loops, and it cannot form a G-quadruplex, which was supported by its Mg2+ -dependent but K+ -independent hemin binding and CD spectroscopy. Isothermal titration calorimetry revealed much higher enthalpy change for the new aptamer, and the best aptamer showed a Kd of 43 nM hemin. Hem1 can also enhance the peroxidase-like activity of hemin. This work demonstrates that aptamers have alternative ways to bind porphyrins allowing selective recognition of different porphyrins.

Pt-Decorated Gold Nanoflares for High-Fidelity Phototheranostics: Reducing Side-Effects and Enhancing Cytotoxicity toward Target Cells.

Functionalized with the Au-S bond, gold nanoflares have emerged as promising candidates for theranostics. However, the presence of intracellular abundantly biothiols compromises the conventional Au-S bond, leading to the unintended release of cargoes and associated side-effects on non-target cells. Additionally, the hypoxic microenvironment in diseased regions limits treatment efficacy, especially in photodynamic therapy. To address these challenges, high-fidelity photodynamic nanoflares constructed on Pt-coated gold nanoparticles (Au@Pt PDNF) were communicated to avoid false-positive therapeutic signals and side-effects caused by biothiol perturbation. Compared with conventional photodynamic gold nanoflares (AuNP PDNF), the Au@Pt PDNF were selectively activated by cancer biomarkers and exhibited high-fidelity phototheranostics while reducing side-effects. Furthermore, the ultrathin Pt-shell catalysis was confirmed to generate oxygen which alleviated hypoxia-related photodynamic resistance and enhanced the antitumor effect. This design might open a new venue to advance theranostics performance and is adaptable to other theranostic nanomaterials by simply adding a Pt shell.

Lowering Entropic Barriers in Triplex DNA Switches Facilitating Biomedical Applications at Physiological pH.

Triplex DNA switches are attractive allosteric tools for engineering smart nanodevices, but their poor triplex-forming capacity at physiological conditions limited the practical applications. To address this challenge, we proposed a low-entropy barrier design to facilitate triplex formation by introducing a hairpin duplex linker into the triplex motif, and the resulting triplex switch was termed as CTNSds. Compared to the conventional clamp-like triplex switch, CTNSds increased the triplex-forming ratio from 30 % to 91 % at pH 7.4 and stabilized the triple-helix structure in FBS and cell lysate. CTNSds was also less sensitive to free-energy disturbances, such as lengthening linkers or mismatches in the triple-helix stem. The CTNSds design was utilized to reversibly isolate CTCs from whole blood, achieving high capture efficiencies (>86 %) at pH 7.4 and release efficiencies (>80 %) at pH 8.0. Our approach broadens the potential applications of DNA switches-based switchable nanodevices, showing great promise in biosensing and biomedicine.

Cross-Binding of Four Adenosine/ATP Aptamers to Caffeine, Theophylline, and Other Methylxanthines.

The classical DNA aptamer for adenosine and ATP was selected twice using ATP as the target in 1995 and 2005, respectively. In 2022, this motif appeared four more times from selections using adenosine, ATP, theophylline, and caffeine as targets, suggesting that this aptamer can also bind methylxanthines. In this work, using thioflavin T fluorescence spectroscopy, this classical DNA aptamer showed Kd values for adenosine, theophylline, and caffeine of 9.5, 101, and 131 µM, respectively, and similar Kd values were obtained using isothermal titration calorimetry. Binding to the methylxanthines was also observed for the newly selected Ade1301 aptamer but not for the Ade1304 aptamer. The RNA aptamer for ATP also had no binding to the methylxanthines. Molecular dynamics simulations were performed using the classical DNA and RNA aptamers based on their NMR structures, and the simulation results were consistent with the experimental observations, explaining the selectivity profiles. This study suggests that a broader range of target analogues need to be tested for aptamers. For the detection of adenosine and ATP, the Ade1304 aptamer is a better choice due to its better selectivity.

Pushing Adenosine and ATP SELEX for DNA Aptamers with Nanomolar Affinity.

The classical DNA aptamer for adenosine and ATP has been the most used small molecule binding aptamer for biosensing, imaging, and DNA nanotechnology. This sequence has recurred multiple times in previous aptamer selections, and all previous selections used a high concentration of ATP as the target. Herein, two separate selections were performed using adenosine and ATP as targets. By pushing the target concentrations down to the low micromolar range, two new aptamers with Kd as low as 230 nM were obtained, showing around 30-fold higher affinity compared to the classical aptamer. The classical aptamer sequence still dominated the library in the early rounds of the selections, but it was suppressed in the later rounds. The new aptamers bind to one target molecule instead of two. Mutation studies confirmed their secondary structures and specific binding. Using the deep sequencing data from the selections, long-standing questions such as the existence of one-site aptamers and mutation distribution in the classical aptamer were addressed. Comparisons were made with previously reported DNA aptamers for ATP. Finally, a strand-displacement biosensor was tested showing selectivity for adenosine and its nucleotides.

Quantitative Comparison of Capture-SELEX, GO-SELEX, and Gold-SELEX for Enrichment of Aptamers.

Since 1990, numerous methods for aptamer selection have been developed, although a quantitative comparison of their sequence enrichment is lacking. In this study, we compared the enrichment factors of three library-immobilization SELEX methods (capture-SELEX, GO-SELEX, and gold-SELEX). We used a spiked library that contained multiple DNA aptamers with different affinities for adenosine. The aptamer separation efficiency was measured using qPCR, and all of the three methods showed a very low DNA release (<1%) in the presence of 100 µM adenosine. Among these, barely any DNA was released from the gold nanoparticles. Deep sequencing was used to compare the enrichment of three aptamers: Ade1301, Ade1304, and the classical aptamer. Enrichment up to 30 to 50-fold was observed only for the capture-SELEX method, whereas the other two methods showed enrichment factors below 1. By blocking the primer-binding regions of the library, GO-SELEX reached up to 14% enrichment. Finally, the enrichment of aptamers based on nonspecific release and target-induced release was discussed, and the advantages of capture-SELEX were rationalized. Taken together, these results indicate that capture-SELEX is a much more efficient method for enriching aptamers.

Cytosine-Rich DNA Binding Insulin Stronger than Guanine-Rich Aptamers: Effect of Aggregation of Insulin for Its Detection.

The detection of insulin is an important analytical task. Previously, guanine-rich DNA was believed to bind insulin, and an insulin aptamer was selected based on a few guanine-rich libraries. Insulin is a unique analyte, and it forms different aggregation states as a function of its concentration and buffer conditions, which may affect the detection of insulin. Herein, using fluorescence polarization assays, three insulin preparation methods were evaluated: direct dissolution, ethylenediaminetetraacetic acid (EDTA) treatment to remove Zn2+, and dissolution in acid followed by neutralization. All the insulin samples containing Zn2+ barely bind to the aptamer DNA, whereas monomers and dimers of insulin with Zn2+ removed were able to bind. Compared to the previously reported aptamer, C-rich DNA showed stronger binding affinities and faster binding kinetics. The sigmoidal binding curves and slow binding kinetics showed that multiple DNA strands and insulin molecules gradually bind, and it took approximately 1 h to reach saturation. This insulin binding was nonspecific, and other tested proteins also can bind to C-rich and G-rich DNA with even strong affinities. These results provide important information on the detection of insulin and further insights into the binding mechanisms between oligomeric insulin and DNA.

Energy Level Engineering in Gold Nanoclusters for Exceptionally Bright NIR Electrochemiluminescence at a Low Trigger Potential.

Electrochemiluminescence (ECL) is a widely used light output mechanism from electrochemical excitation. Understanding the intrinsic essence for ideal ECL generation remains a fundamental challenge. Here, based on the molecular orbital theory, we reported an energy level engineering strategy to regulate the ECL performance by using ligand-protected gold nanoclusters (AuNCs) as luminophores and N,N-diisopropylethylamine (DIPEA) as a coreactant. The energy level matching between the AuNCs and DIPEA effectively promoted their electron transfer reactions, thus improving the excitation efficiency and reducing the trigger potential. Simultaneously, the narrow band gap of the AuNCs further enabled enhanced emission efficiency. Using the energy level engineering theory developed here, a dual-enhanced strategy was proposed, and ß-CD-AuNCs were designed to further verify this mechanism. The ß-CD-AuNCs/DIPEA system resulted in highly stable near-infrared ECL with an unprecedented ECL efficiency (145-fold higher than that of the classic Ru(bpy)32+/tetra-n-butylammonium perchlorate system) and a low trigger potential of 0.48 V. A visual NIR-ECL based on this ECL system was successfully realized by an infrared camera. This work provides an original mechanistic understanding for designing efficient ECL systems, which promises to be a harbinger for broad applicability of this strategy for other ECL systems and ECL sensing platforms.