Insights into In Vivo Environmental Effects on Quantitative Biochemistry in Single Cells.

Biomacromolecules exist and function in a crowded and spatially confined intracellular milieu. Single-cell analysis has been an essential tool for deciphering the molecular mechanisms of cell biology and cellular heterogeneity. However, a sound understanding of in vivo environmental effects on single-cell quantification has not been well established. In this study, via cell mimicking with giant unilamellar vesicles and single-cell analysis by an approach called plasmonic immunosandwich assay (PISA) that we developed previously, we investigated the effects of two in vivo environmental factors, i.e., molecular crowding and spatial confinement, on quantitative biochemistry in the cytoplasm of single cells. We find that molecular crowding greatly affects the biomolecular interactions and immunorecognition-based detection while the effect of spatial confinement in cell-sized space is negligible. Without considering the effect of molecular crowding, the results by PISA were found to be apparently under-quantitated, being only 29.5-50.0% of those by the calibration curve considering the effect of molecular crowding. We further demonstrated that the use of a calibration curve established with standard solutions containing 20% (wt) polyethylene glycol 6000 can well offset the effect of intracellular crowding and thereby provide a simple but accurate calibration for the PISA measurement. Thus, this study not only sheds light on how intracellular environmental factors influence biomolecular interactions and immunorecognition-based single-cell quantification but also provides a simple but effective strategy to make the single-cell analysis more accurate.

Probing Protein 4'-Phosphopantetheinylation in Single Living Cells.

4'-Phosphopantetheinylation (4PPTylation) of proteins, which is derived from the hydrolysis of coenzyme A (CoA), is an essential post-translational modification participating in biosynthetic and metabolic pathways. However, due to the lack of specific recognition ligands as well as the shortage of sensitive analytical tools for single-cell analysis, the in-depth exploration of new cellular functions and mechanisms of protein 4PPTylation has been much hampered. In this study, we rationally engineered CoA-imprinted Raman nanotags for the specific recognition of 4PPTylation and thereby developed a molecularly imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA) for facile probing the 4PPTylation of ALDH1L1 in single cells. The molecularly imprinted nanotags exhibited excellent binding properties, giving a dissociation constant of 10-6 M and cross-reactivity values of less than 10%. The MIP-based PISA enabled the specific and sensitive detection of the level of 4PPTylated ALDH1L1 in single living cells. Particularly, monitoring of the fluctuation of 4PPTylated ALDH1L1 in single cells under simulation by an inhibitor (methotrexate) that acts on a different metabolism pathway was achieved, implying possible crosstalk between two different pathways in folate metabolism. Thus, the imprinted Raman nanotags-PISA provides a promising analytical tool with a single-cell resolution for exploring new functions and elucidating their mechanisms of protein 4PPTylation.

Single-Cell Plasmonic Immunosandwich Assay Reveals the Modulation of Nucleocytoplasmic Localization Fluctuation of ABL1 on Cell Migration.

Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1's shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1's to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein-protein interactions between 14-3-3 and ABL1 modulate ABL1's nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1's nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer.

Dual Biomimetic Recognition-Driven Plasmonic Nanogap-Enhanced Raman Scattering for Ultrasensitive Protein Fingerprinting and Quantitation.

Protein assays with fingerprints and high sensitivity are essential for biomedical research and applications. However, the prevailing methods mainly rely on indirect or labeled immunoassays, failing to provide fingerprint information. Herein, we report a dual biomimetic recognition-driven plasmonic nanogap-enhanced Raman scattering (DBR-PNERS) strategy for ultrasensitive protein fingerprinting and quantitation. A pair of molecularly imprinted nanoantennas were rationally engineered for specifically trapping a target protein into well-defined plasmonic nanogaps through dual-terminal recognition for ultrahigh Raman signal amplification. Meanwhile, a Raman-active small molecule was embedded into the nanoantenna as an internal standard to provide a ratiometric assay for robust quantitation. DBR-PNERS exhibited several significant merits over existing approaches, including fingerprinting, ultrahigh sensitivity, quantitation robustness, speed, sample consumption, and so on. Therefore, it can be a promising tool for a protein assay and holds a great perspective in important applications.

Molecularly Imprinted Nanobeacons Redirect Innate Immune Killing towards Triple Negative Breast Cancer.

Harnessing innate immunity is an appealing strategy for cancer treatment. Herein, we report a new strategy called molecularly imprinted nanobeacons (MINBs) for redirecting innate immune killing towards triple-negative breast cancer (TNBC). The MINBs were molecularly imprinted nanoparticles with the N-epitope of glycoprotein nonmetastatic B (GPNMB) as the template and grafted with plentiful fluorescein moieties as the hapten. The MINBs could tag the TNBC cells via binding with GPNMB and thereby provide navigation for recruiting hapten-specific antibodies. The gathered antibodies could further trigger effective Fc-domain-mediated immune killing towards the tagged cancer cells. In vivo experiments showed that the TNBC growth was significantly inhibited after MINBs treatment by intravenous injection as compared with control groups. This study not only opens a new access for redirecting innate immunity towards TNBC but also paves the way for innate immunity-based therapy of other diseases.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Rationally Screened and Designed ABCG2-Binding Aptamers for Targeting Cancer Stem Cells and Reversing Multidrug Resistance.

The ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2), as an important member of ABC transporters, plays a key role in multidrug resistance (MDR) in cancer and has been widely considered as a marker of cancer stem cells (CSC). Reagents capable of simultaneously targeting ABCG2 and reversing MDR have great clinical application values, but their development is highly challenging. Herein, ABCG2 glycosylated extracellular region-binding aptamers were efficiently screened by a cladded molecularly imprinted polymer (cMIP)-based in vitro screening method and further rationally engineered into cyclic bivalent aptamers. Experiments showed that both the monovalent and cyclic bivalent aptamers could specifically bind ABCG2 and thereby specially target CSC of human colorectal carcinomas (CoCSC), while the latter could effectively reverse MDR in drug-resistant liver cancer cells (HepG2/ADR). Different from currently predominant small molecule inhibitors, the reversal of MDR relied on a different mechanism; the cyclic bivalent aptamers bound the two monomers of ABCG2 dimers simultaneously and thereby blocked the ABCG2-mediated drug-pumping channel, resulting in increased intracellular accumulation of substrate drugs. This study opened a new access to the development of affinity reagents for targeting CSC and reversing MDR, holding great prospects in cancer diagnosis and treatment.

Molecularly Imprinted Nanozymes with Free Substrate Access for Catalyzing the Ligation of ssDNA Sequences.

Nanozymes have attracted wide attention for the unique advantages of low cost, high stability and designability. Molecularly imprinted polymers (MIPs) have demonstrated great potential as a new type of nanozymes due to their excellent specificity and high affinity. However, effective approaches for creating molecularly imprinted nanozymes still remain limited. Herein, reverse microemulsion template docking surface imprinting (RMTD-SI) is reported as a new approach for the rational design and engineering of nanozymes with free substrate access for the ligation of ssDNA sequences. As a proof of the principle, octa-deoxyribonucleotide-imprinted nanoparticles were successfully prepared. Using tetradeoxyribonucleotides and octa-deoxyribonucleotide as substrates, the properties, catalytic activity and behavior of the imprinted nanoparticles were thoroughly investigated. The imprinted nanozyme exhibited an enhanced reaction speed (by up to 41-fold) and good sequence selectivity towards substrate tetra-deoxyribonucleotides. More interestingly, due to the open substrate access, the imprinted nanozyme also allowed the ligation of a ssDNA that fully matched with the imprinted cavity and other ssDNA substrates to form longer sequences, but at the price of substrate selectivity. Thus, this study provides not only a new avenue to the rational design and synthesis of molecularly imprinted nanozymes but also new insights of their catalytic behavior.

Molecularly imprinted and cladded nanoparticles for high-affinity recognition of structurally closed gangliosides.

A new method called reverse microemulsion-confined ganglioside-oriented surface imprinting and cladding (RM-GOSIC) is presented for controllable preparation of nanoscale binders for high-affinity targeting gangliosides. Using GM1a, an affordable ganglioside, as a representative ganglioside target, single-core quantum dot GM1a-imprinted and GM1a-cladded polymer (cMIP) nanoparticles were prepared. The prepared cMIP nanoparticles exhibited extremely high affinity towards GM1a, with dissociation constant at the nanomolar level (3-6 nM). The prepared cMIP nanoparticles also recognized structurally closed gangliosides while their cross-reactivity towards other gangliosides remained low. The potential of the cMIP nanoparticles in biomedical applications was demonstrated by cell and tissue imaging. Thus, this approach opened a new access to the synthesis of high-affinity nanoscale binders for targeting gangliosides.

Molecularly Imprinted and Cladded Nanoparticles Provide Better Phosphorylation Recognition.

Phosphorylation is one of the most frequently occurring post-translation modifications in mammals. Because abnormal protein phosphorylation is related to many diseases, phosphorylation analysis is essential for a sound understanding of protein phosphorylation and its relationship with diseases. Among several types of reagents for phosphorylation recognition, molecularly imprinted polymers (MIPs), as synthetic mimics of antibodies, have exhibited unique strengths that can overcome the drawbacks of biological reagents. However, the performance of current MIPs has remained unideal. Meanwhile, while the currently existing imprinting methods have permitted the production of several material formats, such as crushed particles and mesoporous nanoparticles, a general method allowing for the preparation of monodispersed molecularly imprinted nanoparticles has not been developed yet. Herein, we report a new approach called reverse microemulsion template docking surface imprinting and cladding (RMTD-SIC) for facile preparation of monodispersed imprinted nanoparticles for better phosphorylation recognition. Through rational design and controllable engineering, monodisperse imprinted and cladded nanoparticles specific to general phosphorylation and tyrosine phosphorylation were synthesized, which yield the highest imprinting factors as compared with published studies. The prepared nanomaterials exhibited excellent specificity and affinity, allowing for specific enrichment and improved mass spectrometric identification of target phosphorylated peptides from complex samples containing 100-fold more abundant interfering peptides. Therefore, the RMTD-SIC approach holds great potential for phosphorylation analysis and phosphorylation recognition-based applications.

Efficient Screening of Glycan-Specific Aptamers Using a Glycosylated Peptide as a Scaffold.

Abnormal glycan structures are valuable biomarkers for disease states; the development of glycan-specific binders is thereby significantly important. However, the structural homology and weak immunogenicity of glycans pose major hurdles in the evolution of antibodies, while the poor availability of complex glycans also has extremely hindered the selection of anti-glycan aptamers. Herein, we present a new approach to efficiently screen aptamers toward specific glycans with a complex structure, using a glycosylated peptide as a scaffold. In this method, using peptide-imprinted magnetic nanoparticles (MNPs) as a versatile platform, a glycopeptide tryptically digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection. Alternating positive and negative selection steps were carried out to guide the directed evolution of glycan-binding aptamers. As proof of the principle, the biantennary digalactosylated disialylated N-glycan A2G2S2, against which there have been no antibodies and lectins so far, was employed as the target. With the glycoprotein transferrin as a source of target glycan, two satisfied anti-A2G2S2 aptamers were selected within seven rounds. Since A2G2S2 is upregulated in cancerous liver cells, carboxyfluorescein (FAM)-labeled aptamers were prepared as fluorescent imaging reagents, and successful differentiation of cancerous liver cells over normal liver cells was achieved, which demonstrated the application feasibility of the selected aptamers. This approach obviated a tedious glycan preparation process and allowed favorable expose of the intrinsic flexible conformation of natural glycans. Therefore, it holds great promise for developing glycan-specific aptamers for challenging applications such as cancer targeting.

Controllably Prepared Aptamer-Molecularly Imprinted Polymer Hybrid for High-Specificity and High-Affinity Recognition of Target Proteins.

Molecularly imprinted polymers (MIPs) and aptamers, as effective mimics of antibodies, can overcome only some drawbacks of antibodies. Since they have their own advantages and disadvantages, the combination of MIPs with aptamers could be an ideal solution to produce hybrid alternatives with improved properties and desirable features. Although quite a few attempts have been made in this direction, a facile and controllable approach for the preparation of aptamer-MIP hybrids still remains lacking. Herein, we present a new approach for facile and controllable preparation of aptamer-MIP hybrids for high-specificity and high-affinity recognition toward proteins. An aptamer that can bind the glycoprotein alkaline phosphatase (ALP) with relative weak affinity and specificity was used as a ligand, and controllable oriented surface imprinting was carried out with an in-water self-polymerization system of dopamine. A thin layer of polydopamine was formed to cover the template to an appropriate thickness. After removing the template from the polymer, an aptamer-MIP hybrid with apparently improved affinity and specificity toward ALP was obtained, giving cross-reactivity of 3.2-5.6% and a dissociation constant of 1.5 nM. With this aptamer-MIP hybrid, a plasmonic immunosandwich assay (PISA) was developed. Reliable detection of ALP in human serum by the PISA was demonstrated.

Efficient Mass Spectrometric Dissection of Glycans via Gold Nanoparticle-Assisted in-Source Cation Adduction Dissociation.

Structural identification of glycans is important but remains challenging, for which tandem mass spectrometry has evolved as an indispensable tool. However, it requires additional complex hardware and extra time for ion extraction. Herein, we report a straightforward approach called gold nanoparticles (AuNPs)-assisted in-source cation adduction dissociation (isCAD) for efficient mass spectrometry (MS) dissection of glycans. Although AuNPs have been employed as an inorganic matrix for MALDI MS, this is the first report of AuNP-induced fragmentation. In this approach, AuNPs were employed as an energy absorber for laser ionization as well as a trigger for fragmentation, while residual or deliberately added sodium ions acted as a cationizing agent. The addition of sodium ions induced intensive fragmentation, but the addition of protons suppressed the fragmentation, allowing for facile tuning of the degree of fragmentation. In addition, it was found that larger oligosaccharides and glycans were much easier to fragment as compared with their smaller counterparts, and the use of high-concentration AuNPs effectively suppressed the degree of fragmentation and thereby provided abundant molecular ions. Without any extra hardware and ion extraction, this approach provides a straightforward, cost-efficient and tunable fragmentation for efficient MS dissection of saccharides, including monosaccharides, oligosaccharides, and glycans. Thus, it opens new access to efficient MS dissection of glycans.

Molecularly imprinted and cladded polymers for constructing a portable plasmonic immunoassay for peptides in biofluids.

Using two molecularly imprinted and cladded polymers (cMIPs), an inexpensive, fast and portable plasmonic immuno-sandwich assay (PISA) was rationally developed for high-specificity and ultra-sensitive detection of C-peptide in urine. The dual cMIPs-based PISA allowed healthy individuals to be distinguished from diabetes patients and exhibited several significant merits over existing immunoassays, holding great promise in clinical diagnosis.

Targeted degradation of ABCG2 for reversing multidrug resistance by hypervalent bispecific gold nanoparticle-anchored aptamer chimeras.

Hypervalent bispecific gold nanoparticle-anchored aptamer chimeras (AuNP-APTACs) were designed as a new tool of lysosome-targeting chimeras (LYTACs) for efficient degradation of the ATP-binding cassette, subfamily G, isoform 2 protein (ABCG2) to reverse multidrug resistance (MDR) of cancer cells. The AuNP-APTACs could effectively increase the accumulation of drugs in drug-resistant cancer cells and provide comparable efficacy to small-molecule inhibitors. Thus, this new strategy provides a new way to reverse MDR, holding great promise in cancer therapy.

Hierarchically Structured Molecularly Imprinted Nanotransducers for Truncated HER2-Targeted Photodynamic Therapy of Therapeutic Antibody-Resistant Breast Cancer.

Antibodies have been a mainstream class of therapeutics for clinical treatment of various diseases, especially cancers. However, mutation in cancer cells leads to resistance to therapeutic antibodies, hyperactivity of proliferation of cancer cells, and difficulty in the development of therapeutic antibodies. Herein, we present a strategy termed molecularly imprinted nanotransducer (MINT) for targeted photodynamic therapy (PDT) of mutated cancers. The MINT is a rationally engineered nanocomposite featuring a core of an upconversion nanoparticle, a shell of a thin layer of molecularly imprinted polymer, and a photosensitizer modified on the surface. As a proof-of-principle, truncated HER2 (P95HER2) overexpressed breast cancer, a challenging cancer lacking effective targeted therapeutics, was used as the cancer model. The designed structure, properties, functions, and anticancer efficacy of MINT were systematically investigated and experimentally confirmed. The MINT could not only specifically target P95HER2+ cancer cells in vitro and in vivo but also efficiently transfer the irradiated light and generate excited-state oxygen, resulting in efficient targeted cancer killing. Therefore, the MINT strategy provides a promising therapeutic for targeted PDT of drug-resistant cancers caused by target mutation.

Rational Development of Hypervalent Glycan Shield-Binding Nanoparticles with Broad-Spectrum Inhibition against Fatal Viruses Including SARS-CoV-2 Variants.

Infectious virus diseases, particularly coronavirus disease 2019, have posed a severe threat to public health, whereas the developed therapeutic and prophylactic strategies are seriously challenged by viral evolution and mutation. Therefore, broad-spectrum inhibitors of viruses are highly demanded. Herein, an unprecedented antiviral strategy is reported, targeting the viral glycan shields with hypervalent mannose-binding nanoparticles. The nanoparticles exhibit a unique double-punch mechanism, being capable of not only blocking the virus-receptor interaction but also inducing viral aggregation, thereby allowing for inhibiting the virus entry and facilitating the phagocytosis of viruses. The nanoparticles exhibit potent and broad-spectrum antiviral efficacy to multiple pseudoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its major variants (D614G, N501Y, N439K, Δ69-70, Delta, and Omicron; lentiviruses expressing only the spike proteins), as well as other vital viruses (human immunodeficiency virus 1 and Lassa virus), with apparent EC50 values around the 10-9  m level. Significantly, the broad-spectrum inhibition of authentic viruses of both wild-type SARS-CoV-2 and Delta variants is confirmed. Therefore, this hypervalent glycan-shield targeting strategy opens new access to broad-spectrum viral inhibition.

Rational development of molecularly imprinted nanoparticles for blocking PD-1/PD-L1 axis.

Blocking the PD-1/PD-L1 immune checkpoint has emerged as a promising strategy in cancer immunotherapy, in which monoclonal antibodies are predominately used as inhibitors. Despite their remarkable success, monoclonal antibody-based therapeutics suffer from drawbacks due to the use of antibodies, such as high cost, low stability and high frequency of immune-related adverse effects. Therefore, novel anti-PD-1/PD-L1 therapeutics that can address these issues are of significant importance. Herein, we report a molecularly imprinted polymer (MIP) based PD-1 nano inhibitor for blocking the PD-1/PD-L1 axis. The anti-PD-1 nanoMIP was rationally designed and engineered by epitope imprinting using the N-terminal epitope of PD-1 as the binding site. The anti-PD-1 nanoMIP showed good specificity and high affinity towards PD-1, yielding a disassociation constant at the 10-8 M level, much better than that between PD-1 and PD-L1. Via steric hindrance, this inhibitor could effectively block PD-1/PD-L1 interaction. Besides, it could effectively reactivate T cells and reverse the chemoresistance of tumor cells. Therefore, this present study not only provides a novel and promising immune checkpoint blockade inhibitor but also boosts further development of MIPs for cancer immunotherapy.

Molecularly Imprinted and Cladded Nanotags Enable Specific SERS Bioimaging of Tyrosine Phosphorylation.

Tyrosine phosphorylation is an important post-translational modification of proteins, and its accurate analysis is of vital importance. However, due to limited abundance of tyrosine phosphorylation as well as severe interference of serine/threonine phosphorylation and other phosphate-containing species, approaches that can directly analyse tyrosine phosphorylation on the cell membrane still remain limited. Herein, we report the rational development of molecularly imprinted and cladded Raman nanotags and their successful application in surface enhanced Raman spectroscopy (SERS) imaging of tyrosine phosphorylation on cancerous cells and tissues. The prepared molecularly imprinted and cladded SERS nanotags could specifically recognize phosphotyrosine and thereby allowed for distinguishing phosphotyrosine from other phosphate-containing species on cancerous cells and tissues by SERS imaging. Therefore, the molecularly imprinted and cladded nanotags-based SERS imaging can be a promising tool for tyrosine phosphorylation analysis and tyrosine phosphorylation-related studies, showing great potential for biomedical applications.

Construction of DNA ligase-mimicking nanozymes via molecular imprinting.

Enzyme mimics are of significant importance due to their facile preparation, low cost and stability to rigorous environments. Molecularly imprinted polymers (MIPs) have been important synthetic mimics of enzymes. However, effective strategies for the rational design of enzyme-mimicking MIPs have still remained limited. Herein, we report a new strategy, termed affinity gathering-enhanced coupling and thermal cycling amplification (AGEC-TCA), for the rational design and engineering of molecularly imprinted mesoporous silica nanoparticles (MSNs) that are capable of ligating short ssDNA fragments. This strategy relied on enhancing the effective collision probability via binding substrates into highly favorable orientation by product-imprinted MSNs as well as product release via thermal cycling which enabled successive product amplification. Using modified and natural hexadeoxyribonucleotide as templates, the prepared product-imprinted MSNs exhibited a remarkably enhanced reaction speed (by up to 63-fold) as well as excellent sequence specificity towards substrate trideoxyribonucleotides. Thus, this strategy opened up a new avenue to access enzyme mimics via molecular imprinting.