Corona phase molecular recognition (CoPhMoRe) is a phenomenon whereby a polymer or surfactant corona phase wrapped around a nanoparticle selectively recognizes a particular molecule. The method can potentially generate non-biological, synthetic molecular recognition sites, analogous to antibodies, for a broad range of biomedical applications, including new types of sensors, laboratory and clinical assays, as well as inhibitors and targeted therapeutics. In this work, we utilize near infrared fluorescent single-walled carbon nanotubes (SWNTs) wrapped with specific single stranded DNA sequences to explore the (n,m) chirality dependence of CoPhMoRe. Specific DNA oligonucleotide sequences are known to recognize and interact uniquely with certain (n,m) SWNTs enabling their enrichment in ion exchange chromatography. We explore the CoPhMoRe effect using corona phases constructed from a library of 24 such sequences, screening against a biomolecule panel that includes common neurotransmitters, amino acids, saccharides and riboflavin. Example sequences include (ATT)4, (TAT)4 and (ATTT)3 which recognize (7,5), (6,5) and (8,4) SWNTs, respectively. We find that these recognition sequences indeed form CoPhMoRe phases that are distinct among SWNT chiralities, and appear to pack more densely as to exclude analyte adsorption on the chirality they recognize. These results have encouraging implications for the controlled design of CoPhMoRe phases for biomedical applications.

Effective in silico methods to predict protein corona compositions on engineered nanomaterials (ENMs) could help elucidate the biological outcomes of ENMs in biosystems without the need for conducting lengthy experiments for corona characterization. However, the physicochemical properties of ENMs, used as the descriptors in current modeling methods, are insufficient to represent the complex interactions between ENMs and proteins. Herein, we utilized the fluorescence change (FC) from fluorescamine labeling on a protein, with or without the presence of the ENM, as a novel descriptor of the ENM to build machine learning models for corona formation. FCs were significantly correlated with the abundance of the corresponding proteins in the corona on diverse classes of ENMs, including metal and metal oxides, nanocellulose, and 2D ENMs. Prediction models established by the random forest algorithm using FCs as the ENM descriptors showed better performance than the conventional descriptors, such as ENM size and surface charge, in the prediction of corona formation. Moreover, they were able to predict protein corona formation on ENMs with very heterogeneous properties. We believe this novel descriptor can improve in silico studies of corona formation, leading to a better understanding on the protein adsorption behaviors of diverse ENMs in different biological matrices. Such information is essential for gaining a comprehensive view of how ENMs interact with biological systems in ENM safety and sustainability assessments.

Advancements in optical nanosensor development have enabled the design of sensors using synthetic molecular recognition elements through a recently developed method called Corona Phase Molecular Recognition (CoPhMoRe). The synthetic sensors resulting from these design principles are highly selective for specific analytes, and demonstrate remarkable stability for use under a variety of conditions. An essential element of nanosensor development hinges on the ability to understand the interface between nanoparticles and the associated corona phase surrounding the nanosensor, an environment outside of the range of traditional characterization tools, such as NMR. This review discusses the need for new strategies and instrumentation to study the nanoparticle corona, operating in both in vitro and in vivo environments. Approaches to instrumentation must have the capacity to concurrently monitor nanosensor operation and the molecular changes in the corona phase. A detailed overview of new tools for the understanding of CoPhMoRe mechanisms is provided for future applications.

Temporal and spatial changes in neurotransmitter concentrations are central to information processing in neural networks. Therefore, biosensors for neurotransmitters are essential tools for neuroscience. In this work, we applied a new technique, corona phase molecular recognition (CoPhMoRe), to identify adsorbed polymer phases on fluorescent single-walled carbon nanotubes (SWCNTs) that allow for the selective detection of specific neurotransmitters, including dopamine. We functionalized and suspended SWCNTs with a library of different polymers (n = 30) containing phospholipids, nucleic acids, and amphiphilic polymers to study how neurotransmitters modulate the resulting band gap, near-infrared (nIR) fluorescence of the SWCNT. We identified several corona phases that enable the selective detection of neurotransmitters. Catecholamines such as dopamine increased the fluorescence of specific single-stranded DNA- and RNA-wrapped SWCNTs by 58-80% upon addition of 100 μM dopamine depending on the SWCNT chirality (n,m). In solution, the limit of detection was 11 nM [K(d) = 433 nM for (GT)15 DNA-wrapped SWCNTs]. Mechanistic studies revealed that this turn-on response is due to an increase in fluorescence quantum yield and not covalent modification of the SWCNT or scavenging of reactive oxygen species. When immobilized on a surface, the fluorescence intensity of a single DNA- or RNA-wrapped SWCNT is enhanced by a factor of up to 5.39 ± 1.44, whereby fluorescence signals are reversible. Our findings indicate that certain DNA/RNA coronae act as conformational switches on SWCNTs, which reversibly modulate the SWCNT fluorescence. These findings suggest that our polymer-SWCNT constructs can act as fluorescent neurotransmitter sensors in the tissue-compatible nIR optical window, which may find applications in neuroscience.

Corona phase molecular recognition (CoPhMoRe) uses a heteropolymer adsorbed onto and templated by a nanoparticle surface to recognize a specific target analyte. This method has not yet been extended to macromolecular analytes, including proteins. Herein we develop a variant of a CoPhMoRe screening procedure of single-walled carbon nanotubes (SWCNT) and use it against a panel of human blood proteins, revealing a specific corona phase that recognizes fibrinogen with high selectivity. In response to fibrinogen binding, SWCNT fluorescence decreases by >80% at saturation. Sequential binding of the three fibrinogen nodules is suggested by selective fluorescence quenching by isolated sub-domains and validated by the quenching kinetics. The fibrinogen recognition also occurs in serum environment, at the clinically relevant fibrinogen concentrations in the human blood. These results open new avenues for synthetic, non-biological antibody analogues that recognize biological macromolecules, and hold great promise for medical and clinical applications.

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Protein A is often used for the purification and detection of antibodies such as immunoglobulin G (IgG) because of its quadrivalent domains that bind to the Fc region of these macromolecules. However, the kinetics and thermodynamics of the binding to many sensor surfaces have eluded mechanistic description due to complexities associated with multivalent interactions. In this work, we use a near-infrared (nIR) fluorescent single-walled carbon nanotube sensor array to obtain the kinetics of IgG binding to protein A, immobilized using a chelated Cu(2+)/His-tag chemistry to hydrogel dispersed sensors. A bivalent binding mechanism is able to describe the concentration dependence of the effective dissociation constant, KD,eff, which varies from 100 pM to 1 μM for IgG concentrations from 1 ng mL(-1) to 100 μg mL(-1), respectively. The mechanism is shown to describe the unusual concentration-dependent scaling demonstrated by other sensor platforms in the literature as well, and a comparison is made between resulting parameters. For comparison, we contrast IgG binding with that of human growth hormone (hGH) to its receptor (hGH-R) which displays an invariant dissociation constant at KD = 9 μM. These results should aid in the use of protein A and other recognition elements in a variety of sensor types.

Applications of reduced graphene oxide (rGO) in many different areas have been gradually increasing owing to its unique physicochemical characteristics, demanding more understanding of their biological impacts. Herein, we assessed the toxicological effects of rGO in mammary epithelial cells. Because the as-synthesized rGO was dissolved in sodium cholate to maintain a stable aqueous dispersion, we hypothesize that changing the cholate concentration in the dispersion may alter the surface property of rGO and subsequently affect its cellular toxicity. Thus, four types of rGO were prepared and compared: rGO dispersed in 4 and 2 mg/mL sodium cholate, labeled as rGO and concentrated-rGO (c-rGO), respectively, and rGO and c-rGO coated with a protein corona through 1 h incubation in culture media, correspondingly named pro-rGO and pro-c-rGO. Notably, c-rGO and pro-c-rGO exhibited higher toxicity than rGO and pro-rGO and also caused higher reactive oxygen species production, more lipid membrane peroxidation, and more significant disruption of mitochondrial-based ATP synthesis. In all toxicological assessments, pro-c-rGO induced more severe adverse impacts than c-rGO. Further examination of the material surface, protein adsorption, and cellular uptake showed that the surface of c-rGO was coated with a lower content of surfactant and adsorbed more proteins, which may result in the higher cellular uptake observed with pro-c-rGO than pro-rGO. Several proteins involved in cellular redox mediation were also more enriched in pro-c-rGO. These results support the strong correlation between dispersant coating and corona formation and their subsequent cellular impacts. Future studies in this direction could reveal a deeper understanding of the correlation and the specific cellular pathways involved and help gain knowledge on how the toxicity of rGO could be modulated through surface modification, guiding the sustainable applications of rGO.