Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Spin-enhanced nanodiamond biosensing for ultrasensitive diagnostics.

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications1. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness2, low cost3 and selective manipulation of their emission4. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity5 and frequency-domain analysis6 to separate the signal from background autofluorescence7, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10-19 molar for a biotin-avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Microfluidic measurement of the size and shape of lipid-anchored proteins.

The surface of a cell is crowded with membrane proteins. The size, shape, density, and mobility of extracellular surface proteins mediate cell surface accessibility to external molecules, viral particles, and other cells. However, predicting these qualities is not always straightforward, even when protein structures are known. We previously developed an experimental method for measuring flow-driven lateral transport of neutravidin bound to biotinylated lipids in supported lipid bilayers. Here, we use this method to detect hydrodynamic force applied to a series of lipid-anchored proteins with increasing size. We find that the measured force reflects both protein size and shape, making it possible to distinguish these features of intact, folded proteins in their undisturbed orientation and proximity to the lipid membrane. In addition, our results demonstrate that individual proteins are transported large distances by flow forces on the order of femtoNewtons, similar in magnitude to the shear forces resulting from blood circulation or from the swimming motion of microorganisms. Similar protein transport across living cells by hydrodynamic force may contribute to biological flow sensing.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

Development of sterile platform for quantification of extracellular analytes via single walled carbon nanotubes.

Progress has been made studying cell-cell signaling communication processes. However, due to limitations of current sensors on time and spatial resolution, the role of many extracellular analytes is still unknown. A single walled carbon nanotube (SWNT) platform was previously developed based on the avidin-biotin immobilization of SWNT to a glass substrate. The SWNT platform provides real time feedback about analyte concentration and has a high concentration of evenly distributed sensors, both of which are essential for the study of extracellular analytes. Unfortunately, this initial SWNT platform is synthesized through unsterile conditions and cannot be sterilized post-production due to the delicate nature of the sensors, making it unsuitable for in vitro work. Herein the multiple-step process for SWNT immobilization is modified and the platform's biocompatibility is assessed in terms of sterility, cytotoxicity, cell proliferation, and cell morphology through comparison with non-sensors controls. The results demonstrate the SWNT platform's sterility and lack of toxicity over 72 h. The proliferation rate and morphology profiles for cells growing on the SWNT platform are similar to those grown on tissue culture substrates. This novel nano-sensor platform preserves cell health and cell functionality over time, offering opportunities to study extracellular analytes gradients in cellular communication.

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

Real-time monitoring of voltage-responsive biomolecular binding onto electro-switchable surfaces.

Voltage-responsive biosensors capable of monitoring real-time adsorption behavior of biological analytes onto electroactive surfaces offer attractive strategies for disease detection, separations, and other adsorption-dependent analytical techniques. Adsorption of biological analytes onto electrically switchable surfaces can be modelled using neutravidin and biotin. Here, we report self-assembled monolayers formed from voltage-switchable biotinylated molecules on gold surfaces with tunable sensitivity to neutravidin in response to applied voltages. By using electrochemical quartz crystal microbalance (EQCM), we demonstrated real-time switchable behavior of these bio-surfaces and investigate the range of sensitivity by varying the potential of the same surfaces from -400 mV to open circuit potential (+155 mV) to +300 mV. We compared the tunability of the mixed surfaces to bare Au surfaces, voltage inert surfaces, and switchable biotinylated surfaces. Our results indicate that quartz crystal microbalance allows real-time changes in analyte binding behavior, which enabled observing the evolution of neutravidin sensitivity as the applied voltage was shifted. EQCM could in principle be used in kinetic studies or to optimize voltage-switchable surfaces in adsorption-based diagnostics.

Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

A novel mertansine conjugate for acid-reversible targeted drug delivery validated through the Avidin-Nucleic-Acid-NanoASsembly platform.

In targeted cancer therapy, antibody-drug-conjugates using mertansine (DM1)-based cytotoxic compounds rely on covalent bonds for drug conjugation. Consequently, the cytotoxic DM1 derivative released upon their proteolytic digestion is up to 1000-fold less potent than DM1 and lacks a bystander effect. To overcome these limitations, we developed a DM1 derivative (keto-DM1) suitable for bioconjugation through an acid-reversible hydrazone bond. Its acid-reversible hydrazone conjugate with biotin (B-Hz-DM1) was generated and tested for efficacy using the cetuximab-targeted Avidin-Nucleic-Acid-NanoASsembly (ANANAS) nanoparticle (NP) platform. NP-tethered B-Hz-DM1 is stable at neutral pH and releases its active moiety only in endosome/lysosome mimicking acidic pH. In vitro, the NP/Cetux/B-Hz-DM1 assembly showed high potency on MDA-MB231 breast cancer cells. In vivo both B-Hz-DM1 and NP/Cetux/B-Hz-DM1 reduced tumor growth. A significantly major effect was exerted by the nanoformulation, associated with an increased in situ tumor cell death. Keto-DM1 is a promising acid-reversible mertansine derivative for targeted delivery in cancer therapy.

Expanding the Hydrophobic Cavity Surface of Azocalix[4]arene to Enable Biotin/Avidin Affinity with Controlled Release.

The development of artificial receptors that combine ultrahigh-affinity binding and controllable release for active guests holds significant importance in biomedical applications. On one hand, a complex with an exceedingly high binding affinity can resist unwanted dissociation induced by dilution effect and complex interferents within physiological environments. On the other hand, stimulus-responsive release of the guest is essential for precisely activating its function. In this context, we expanded hydrophobic cavity surface of a hypoxia-responsive azocalix[4]arene, affording Naph-SAC4A. This modification significantly enhanced its aqueous binding affinity to 1013 M-1, akin to the naturally occurring strongest recognition pair, biotin/(strept-)avidin. Consequently, Naph-SAC4A emerges as the first artificial receptor to simultaneously integrate ultrahigh recognition affinity and actively controllable release. The markedly enhanced affinity not only improved Naph-SAC4A's sensitivity in detecting rocuronium bromide in serum, but also refined the precision of hypoxia-responsive doxorubicin delivery at the cellular level, demonstrating its immense potential for diverse practical applications.

The avidin-theophylline complex: A structural and computational study.

The interaction between avidin and its counterpart biotin is one of central importance in biology and has been reproposed and studied at length. However, the binding pocket of avidin is prone to promiscuous binding, able to accommodate even non-biotinylated ligands. Comprehending the factors that distinguish the extremely strong interaction with biotin to other ligands is an important step to fully picture the thermodynamics of these low-affinity complexes. Here, we present the complex between chicken white egg avidin and theophylline (TEP), the xanthine derivative used in the therapy of asthma. In the crystal structure, TEP lies in the biotin-binding pocket with the same orientation and planarity of the aromatic ring of 8-oxodeoxyguanosine. Indeed, its affinity for avidin measured by isothermal titration calorimetry is in the same µM range as those obtained for the previously characterized nucleoside derivatives. By the use of molecular dynamic simulations, we have investigated the most important intermolecular interactions occurring in the avidin-TEP binding pocket and compared them with those obtained for the avidin 8-oxodeoxyguanosine and avidin-biotin complexes. These results testify the capability of avidin to complex purely aromatic molecules.

Protein-Scaffolded DNA Nanostructures for Imaging of Apurinic/Apyrimidinic Endonuclease 1 Activity in Live Cells.

Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.

PD-L1 ImmunoPET on the basis of Avidin/Biotin pre-targeted cancer imaging.

This study aimed to establish the radio-immune imaging protocol on the basis of Avidin/Biotin system. The programmed death-ligand 1 (PD-L1) antibody (Atezolizumab) was employed as the primary molecule in targeting PD-L1, and the two-step strategy, consisting of the first injection of Avidin-conjugated PD-L1 monoclonal antibody (Atezolizumab) and the second injection of 7.4 MBq 68Ga-Biotin with a 60 h interval, was then verified on the colon cancer-bearing mice. PET imaging was performed at 30, 90, 180 min to measure the standard uptake value and tumor to liver ratios. Cellular binding experiments and in vivo distribution showed that the conjugation of Avidin did not affect the affinity of Atezolizumab to PD-L1 antigen. Biotin was radio-labeled with 68Ga with radiolabeling efficiency of 70.5 ± 3.5% and purification was needed to increase the radiochemical purity. For PD-L1-positive tumors, SUVmax was 0.38 ± 0.06 in the Avidin-Atezolizumab pre-treated mice at 90 min; the tumor/liver ratios of pre-targeting group were 1.06 ± 0.19 and 0.97 ± 0.16 at 30 and 90 min, while the absence of pre-treatment of Avidin was of the lower ratios as 0.88 ± 0.01 and 0.54 ± 0.11 when 68Ga-Biotin served as the radiopharmaceutical as well. In conclusion, pre-targeting immunoPET strategy can elevate the target-to-nontarget ratio, decrease the blood background and shorten the interval between injection of radiopharmaceuticals and PET scan, providing a highly PD-L1-specific and sensitive imaging method for the detection of tumorous immune micro-environment.

Intra-articular kinetics of a cartilage targeting cationic PEGylated protein for applications in drug delivery.

OBJECTIVES: Cartilage targeting cationic glycoprotein Avidin was PEGylated to synthesize a multi-arm Avidin (mAv) nano-construct with high drug loading content. Here we investigate mAv biodistribution and kinetics over a 7-day period following intra-articular (IA) administration in rat knee joints. METHODS: Labeled mAv was injected into healthy rat knees, and joint tissues (articular cartilage, menisci, ligaments, tendons, fat pad) were harvested following sacrifice at 6 h, 1, 4 and 7 days. Its IA biodistribution and retention were measured using fluorescence microscopy. Tissue localization was compared in young vs old rats by immunohistochemistry. mAv chondrotoxicity and immune response were evaluated to determine safe carrier dose limits. RESULTS: mAv penetrated through the full thickness of rat cartilage and other joint tissues within 6 h, remaining detectable within most joint tissues over 7 days. Intra-tissue uptake correlated strongly with tissue GAG concentration, confirming the dominant role of electrostatic interactions between positively charged mAv and the negatively charged aggrecan proteoglycans. mAv was uptaken by chondrocytes and also penetrated the osteocyte lacuno-canalicular system of peri-articular bone in both young and old rats. mAv did not cause cytotoxicity at concentrations up to 300 µM but elicited a dose dependent immunogenic response. CONCLUSIONS: mAv's ability to target a variety of joint tissues, chondrocytes, and peri-articular osteocytes without sequestration in synovial fluid makes it a versatile carrier for delivering a wide range of drugs for treating a broad class of musculoskeletal diseases. Drugs can be conjugated using simple aqueous based avidin-biotin reaction, supporting its clinical prospects.

Sustained intra-cartilage delivery of interleukin-1 receptor antagonist using cationic peptide and protein-based carriers.

OBJECTIVE: Blocking the interleukin-1 (IL-1) catabolic cascade following joint trauma can be achieved using its receptor antagonist, IL-1Ra. However, its clinical translation for osteoarthritis therapy has been unsuccessful due to its rapid joint clearance and lack of targeting and penetration into deep cartilage layers at therapeutic concentrations. Here, we target the high negative charge of cartilage aggrecan-glycosaminoglycans (GAGs) by attaching cationic carriers to IL-1Ra. IL-1Ra was conjugated to the cartilage targeting glycoprotein, Avidin, and a short length optimally charged cationic peptide carrier (CPC+14). It is hypothesized that electro-diffusive transport and binding properties of IL-1Ra-Avidin and IL-1Ra-CPC+14 will create intra-cartilage depots of IL-1Ra, resulting in long-term suppression of IL-1 catabolism with only a single administration. DESIGN: IL-1Ra was conjugated to Avidin or CPC+14 using site specific maleimide linkers, and confirmed using gel electrophoresis, high-performance liquid chromatography (HPLC), and mass spectrometry. Intra-cartilage transport and retention of conjugates was compared with native IL-1Ra. Attenuation of IL-1 catabolic signaling with one-time dose of IL-1Ra-CPC+14 and IL-1Ra-Avidin was assessed over 16 days using IL-1α challenged bovine cartilage and compared with unmodified IL-1Ra. RESULTS: Positively charged IL-1Ra penetrated through the full-thickness of cartilage, creating a drug depot. A single dose of unmodified IL-1Ra was not sufficient to attenuate IL-1-induced cartilage deterioration over 16 days. However, when delivered using Avidin, and to a greater extent CPC+14, IL-1Ra significantly suppressed cytokine induced GAG loss and nitrite release while improving cell metabolism and viability. CONCLUSION: Charge-based cartilage targeting drug delivery systems hold promise as they can enable long-term therapeutic benefit with only a single dose.

Quantifying and Modulating Protein Encapsulation in Guanosine-Based Supramolecular Particles.

The encapsulation of proteins is an effective way to preserve their structure and enhance their function. One exciting possibility is adjusting the protective agent to match the specific protein's characteristics to influence its properties. In a recent study, we developed a flow cytometry-based method to quantify the encapsulation of small-molecule dyes in colloidal particles made from guanosine derivatives (supramolecular hacky sacks (SHS) particles). We aimed to determine whether this method could quantify protein encapsulation and track changes and if the particles could be tuned to bind to specific proteins. Our results showed that fluorescein isothiocyanate (FITC)-labeled proteins had apparent association constants in the micromolar range with hydrophobicity as the dominant factor enhancing the affinities. Confocal laser scanning microscopy (CLSM) imaging supported these results and provided additional information about the protein distribution within the particles. We also tested the feasibility of tuning the avidin affinity (AVI) for SHS particles with a biotin ligand. We found that increasing the amount of biotin initially enhanced AVI binding, but then reached saturation, which we hypothesize results from noncovalent cross-linking caused by strong biotin/AVI interactions. CLSM images showed that the linker also impacted the AVI distribution within the particles. Our strategy provides an advantage over other methods for quantifying protein encapsulation by being suitable for high-throughput analysis with high reproducibility. We anticipate that future efforts to use lower-affinity ligands would result in better strategies for modulating protein affinity for drug delivery applications.

Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.

PURPOSE: Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the ß-emitter 90Y linked to DOTA-biotin-avidin ([90Y]DBA) to deliver short-range radiation against non-muscle invasive bladder cancer (NMIBC). MATERIAL AND METHODS: Image-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with the 68Ga and dynamic microPET imaging following intravesical infusion of [68Ga]DBA for up to 4 h and post-necropsy γ-counting of organs. The antitumor activity of [90Y]DBA was investigated using an orthotopic MB49 murine bladder cancer model. Mice were injected with luciferase-expressing MB49 cells and treated via intravesical administration with 9.2 MBq of [90Y]DBA or unlabeled DBA 3 days after the tumor implantation. Bioluminescence imaging was conducted after tumor implantation to monitor the bladder tumor growth. In addition, we investigated the effects of [90Y]DBA radiation on urothelial histology with immunohistochemistry analysis of bladder morphology. RESULTS: Our results demonstrated that DBA is contained in the bladder for up to 4 h after intravesical infusion. A single dose of [90Y]DBA radiation treatment significantly reduced growth of MB49 bladder carcinoma. Attaching 90Y-DOTA-biotin to avidin prevents its re-absorption into the blood and distribution throughout the rest of the body. Furthermore, immunohistochemistry demonstrated that [90Y]DBA radiation treatment did not cause short-term damage to urothelium at day 10, which appeared similar to the normal urothelium of healthy mice. CONCLUSION: Our data demonstrates the potential of intravesical [90Y]DBA as a treatment for non-muscle invasive bladder cancer.

Surface imprinted bio-nanocomposites for affinity separation of a cellular DNA repair protein.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and "interactomes" of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio-nanocomposite with antibody-like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin-modified surface of silica-coated magnetic nanoparticles, we first added 3-aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2-acrylamido-2-methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG-NH2 ). The resulting molecularly imprinted polymer-based bio-nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio-nanocomposite with high activity. The bio-nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.

Enzymatic Janus Liposome Micromotors.

A liposome-based micromotor system that utilizes regional enzymatic conversion and gas generation to achieve directional motion in water is presented. Constituted mainly of a low-melting lipid and a high-melting lipid together with cholesterol, these liposomes maintain stable Janus configuration at room temperature as a result of lipid liquid-liquid phase separation. Local placement of enzymes such as horseradish peroxidase is realized via affinity binding between avidin and biotin, the latter as a lipid conjugate sorted specifically into one domain of these Janus liposomes as a minor component. In the presence of the substrate, hydrogen peroxide, these enzyme-decorated Janus liposomes undergo directional motion, yielding velocities exceeding thermal diffusion by three folds in some cases. Experimental details on liposome size control, motor assembly, and substrate distribution are presented; effects of key experimental factors on liposome motion, such as substrate concentration and liposome Janus ratio, are also examined. This work thus provides a viable approach to building asymmetrical lipid-assembled, enzyme-attached colloids and, in addition, stresses the importance of asymmetry in achieving particle directional motion.