Logic-gated engineered cells are an emerging therapeutic modality that can take advantage of molecular profiles to focus medical interventions on specific tissues in the body. However, the increased complexity of these engineered systems may pose a challenge for prediction and optimization of their behavior. Here we describe the design and testing of a flow cytometry-based screening system to rapidly select functional inhibitory receptors from a pooled library of candidate constructs. In proof-of-concept experiments, this approach identifies inhibitory receptors that can operate as NOT gates when paired with activating receptors. The method may be used to generate large datasets to train machine learning models to better predict and optimize the function of logic-gated cell therapeutics.

A chemical reaction and its reaction environment are intrinsically linked, especially within the confines of narrow cellular spaces. Traditional models of chemical reactions often use differential equations with concentration as the primary variable, neglecting the density heterogeneity in the solution and the interaction between the reaction and its environment. We model the interaction between a chemical reaction and its environment within a geometrically confined space, such as inside a cell, by representing the environment through the size of molecular clusters. In the absence of fluctuations, the interplay between cluster size changes and the activation and inactivation of molecules induces oscillations. However, in unstable environments, the system reaches a fluctuating steady state. When an enzyme is introduced to this steady state, oscillations akin to action potential spike trains emerge. We examine the behavior of these spike trains and demonstrate that they can be used to implement logic gates. We discuss the oscillations and computations that arise from the interaction between a chemical reaction and its environment, exploring their potential for contributing to chemical intelligence.

The low solubility of chitosan (CS) imposes adverse effects on its application. In this work, one of the aims is to improve the water solubility of CS. By introducing water-soluble side chains to CS, this aim was achieved. Besides, fluorescent moieties were incorporated into the side chains, the fluorescent copolymers were endowed with Cr3+ and Cu2+ ions recognition ability. Firstly, a reversible addition-fragmentation chain transfer polymerization (RAFT) reagent with naphthalimide units and CC groups was prepared. Water-soluble monomer methyl acrylic acid (MAA) was employed in the RAFT polymerization. Thus, water-soluble polymer with fluorescent unit and -C ≡ C on both ends of the polymer was obtained. They were introduced into CS, and the CS-based fluorescent copolymers were obtained eventually. The amount of MAA introduced could be tuned to obtain three side chains of different lengths. It was found that the more MAA was introduced, the better the solubility of CS-TP was. The detection limits (LOD) of Cr3+ and Cu2+ were 44.6 nM and 54.5 nM, respectively. The detection of Cr3+ and Cu2+ ions is further combined with a mobile APP to realize real-time, portable, and visual detection. And the application in the logic gate, a new detection platform, is prepared.

Dimethylaniline-substituted aza-BODIPY dyes (DA, DM, DP) were designed and synthesized aiming for ion detection. The Zn2+ recognition ability was found in all compounds and the binding mechanism was possibly via dimethylaniline sites linked to the aza-BODIPY core. Upon Zn2+ addition, the new absorption band and the color change occurred due to the altered charge transfer of the adducts. The custom-made colorimeter was successfully integrated into the dye\'s application, demonstrating a good linear relationship between resistance values and Zn2+ concentration. The chromophore test strips were fabricated and exhibited distinct color changes upon aqueous Zn2+ exposure. The compound DA also exhibits logical behavior with DA-Zn2+-Cu2+ system. In terms of environmental hazards, the compounds exhibited no adverse effect on Pseudomonas putida at the concentration level of 0.2 mg/mL. These findings indicated that all synthesized aza-BODIPYs might be suitable for chemosensor probes for Zn2+ detection with possibly low environmental risk.

Molecular beacons (MBs) based on hairpin-shaped oligonucleotides are captivating owing to their capability to enable effective real-time detection of cytosolic mRNA in living cells. However, DNase in the nucleus and lysosome could induce the degradation of oligonucleotides in MBs, leading to the generation of false-positive signals. Herein, a graphene oxide (GO) nanosheet was applied as a nanocarrier for MBs to greatly enhance the anti-interference of the easily designed nanoprobe. Advantageously, the absorption capacity of GO for MBs increased with the decrease in pH values, providing the MB-GO nanoprobe with the ability to detect the expression of cytosolic Ki-67 mRNA without interference from DNase Ⅱ in lysosomes. Moreover, the size of GO nanosheets was considerably higher than that of the nuclear pore complex (NPC), which prevented nanoprobes from transition through the NPCs, thereby avoiding the generation of false-positive signals in the nucleus. Altogether, the present work affords a convenient approach for the successful detection of Ki-67 mRNA expression in the cytosol without interference from DNase Ⅰ/Ⅱ in the nucleus/lysosome, which may be potentially further applied for the detection of other cytosolic RNAs.

A ninhydrin-based colorimetric chemosensor (LH) was synthesized using 3-hydroxy-2-naphthoic hydrazide and 11H-indeno[1,2-b]quinoxalin-11-one. It was characterized by spectroscopic and single crystal X-ray diffraction techniques. In a semi-aqueous (MeOH/HEPES) system, LH displayed a characteristic chromogenic change from colorless to yellow upon adding Cu2+ ion, with the appearance of a new peak at λmax = 460 nm. A 1:1 binding stoichiometry between LH and Cu2+ ion has been found, with LOD = 2.3 μM (145 ppb) and LOQ = 8 μM (504 ppb). Based on experimental results the formula of [Cu(L)Cl(H2O)2] (1) was assigned and this in-situ generated 1 was found to exhibit a discoloration of upon gradual addition of cysteine (LOD = 60 nM) as well as ATP (LOD = 130 nM) having 1:2 and 1:1 stoichiometry respectively. The LH was useful for recognition of Cu2+ ion in real water samples and on filter paper strips. A two-input-two-output logic gate circuitry was also constructed by employing 1 and cysteine. The DFT/TDDFT calculations performed on LH and 1 were consistent with experimental findings. The binding affinity of LH towards HSA and BSA were determined with HSA having greater affinity than BSA, which was also supported by theoretical calculations.

Understanding the transport of nanoparticles from and within hydrogels is a key issue for the design of nanocomposite hydrogels for drug delivery systems and tissue engineering. To investigate the translocation of nanocarriers from and within hydrogel networks triggered by changes of temperature, ultrasmall (8 nm) and small (80 nm) silica nanocapsules are embedded in temperature-responsive hydrogels and non-responsive hydrogels. The ultrasmall silica nanocapsules are released from temperature-responsive hydrogels to water or transported to other hydrogels upon direct activation by heating or indirect activation by Joule heating; while, they are not released from non-responsive hydrogel. Programmable transport of nanocarriers from and in hydrogels provides insights for the development of complex biomedical devices and soft robotics.

The development of sensitive and efficient analytical methods for multiple biomarkers is crucial for cancer screening at early stage. MicroRNAs (miRNAs) are a kind of biomarkers with diagnostic potential for cancer. However, the ultrasensitive and logical analysis of multiple miRNAs with simple operation still faces some challenges. Herein, a photonic crystal (PC)-enhanced fluorescence biosensor with logic gate operation based on one-pot cascade amplification DNA circuit was developed for enzyme-free and ultrasensitive analysis of two cancer-related miRNAs. The fluorescence biosensor was performed by biochemical recognition amplification module (BCRAM) and physical enhancement module (PEM) to achieve logical and sensitive detection. In the BCRAM, one-pot cascade amplification circuit consisted of the upstream parallel entropy-driven circuit (EDC) and the downstream shared catalytic hairpin assembly (CHA). The input of target miRNA would trigger each corresponding EDC, and the parallel EDCs released the same R strand for triggering subsequent CHA; thus, the OR logic gate was obtained with minimization of design and operation. In the PEM, photonic crystal (PC) array was prepared easily for specifically enhancing the fluorescence output from BCRAM by the optical modulation capabilities; meanwhile, the high-throughput signal readout was achieved by microplate analyzer. Benefiting from the integrated advantages of two modules, the proposed biosensor achieved ultrasensitive detection of two miRNAs with easy logic gate operation, obtaining the LODs of 8.6 fM and 6.7 fM under isothermal and enzyme-free conditions. Hence, the biosensor has the advantages of high sensitivity, easy operation, multiplex and high-throughput analysis, showing great potential for cancer screening at early stage.

A highly efficient fluorescent sensor (S-DAC) was easily created by functionalizing the SBA-15 surface with N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane followed by the covalent attachment of 7-diethylamino 3-acetyl coumarin (DAC). This chemosensor (S-DAC) demonstrates selective and sensitive recognition of Fe3+ and Hg2+ in water-based solutions, with detection limits of 0.28 × 10-9 M and 0.2 × 10-9 M for Hg2+ and Fe3+, respectively. The sensor\'s fluorescence characteristics were examined in the presence of various metal ions, revealing a decrease in fluorescence intensity upon adding Fe3+ or Hg2+ ions at an emission wavelength of 400 nm. This sensor was also able to detect ferric and mercury ions in spinach and tuna fish. The quenching mechanism of S-DAC was investigated using UV-vis spectroscopy, which confirmed a static-type mechanism for fluorescence quenching. Moreovre, the decrease in fluorescence intensity caused by mercury and ferric ions can be reversed using trisodium citrate dihydrate and EDTA as masking agents, respectively. As a result, a circuit logic gate was designed using Hg2+, Fe3+, trisodium citrate dihydrate, and EDTA as inputs and the quenched fluorescence emission as the output.

Clinical diagnosis of ovarian cancer lacks high accuracy due to the weak selection of specific biomarkers along with the circumstance biomarkers localization. Clustering analysis of proteins transported on exosomes enables a more precise screening of effective biomarkers. Herein, through bioinformatics analysis of ovarian cancer and exosome proteomes, two coexpressed proteins, EpCAM and CD24, specifically enriched, were identified, together with the development of an as-derived dual-aptamer targeted exosome-based strategy for ovarian cancer screening. In brief, a DNA ternary polymer with aptamers targeting EpCAM and CD24 was designed to present a logic gate reaction upon recognizing ovarian cancer exosomes, triggering a rolling circle amplification chemiluminescent signal. A dynamic detection range of 6 orders of magnitude was achieved by quantifying exosomes. Moreover, for clinical samples, this strategy could accurately differentiate exosomes from healthy persons, other cancer patients, and ovarian cancer patients, enabling promising in situ detection. By accurately selecting biomarkers and constructing a dual-targeted exosomal protein detection strategy, the limitation of insufficient specificity of traditional protein markers was circumvented. This work contributed to the development of exosome-based prognosis monitoring in ovarian cancer through the identification of disease-specific exosome protein markers.