Zhiwen Tang

Protein-based nanomedicine platforms for drug delivery.

Protein-based nanomedicine platforms for drug delivery comprise naturally self-assembled protein subunits of the same protein or a combination of proteins making up a complete system. They are ideal for drug-delivery platforms due to their biocompatibility and biodegradability coupled with low toxicity. A variety of proteins have been used and characterized for drug-delivery systems, including the ferritin/apoferritin protein cage, plant-derived viral capsids, the small Heat shock protein (sHsp) cage, albumin, soy and whey protein, collagen, and gelatin. There are many different types and shapes that have been prepared to deliver drug molecules using protein-based platforms, including various protein cages, microspheres, nanoparticles, hydrogels, films, minirods, and minipellets. The protein cage is the most newly developed biomaterial for drug delivery and therapeutic applications. The uniform size, multifunctionality, and biodegradability push it to the frontier of drug delivery. In this Review, the recent strategic development of drug delivery is discussed with emphasis on polymer-based, especially protein-based, nanomedicine platforms for drug delivery. The advantages and disadvantages are also discussed for each type of protein-based drug-delivery system.

Constraint of DNA on Functionalized Graphene Improves its Biostability and Specificity

The single-stranded DNA constrained on graphene surface is effectively protected from enzymatic cleavage by DNase I. The anisotropy, fluorescence, NMR, and CD studies suggest that the single-stranded DNA is promptly adsorbed onto graphene forming strong molecular interactions. Furthermore, the constraint of DNA probe on graphene improves the specificity of its response to complementary DNA. These findings will promote the further application of graphene in biotechnology and biomedical fields.

Rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow test strip.

A portable fluorescence biosensor with rapid and ultrasensitive response for protein biomarker has been built up with quantum dots and a lateral flow test strip. The superior signal brightness and high photostability of quantum dots are combined with the promising advantages of a lateral flow test strip and result in high sensitivity and selectivity and speed for protein detection. Nitrated ceruloplasmin, a significant biomarker for cardiovascular disease, lung cancer, and stress response to smoking, was used as model protein biomarker to demonstrate the good performances of this proposed quantum dot-based lateral flow test strip. Quantitative detection of nitrated ceruloplasmin was realized by recording the fluorescence intensity of quantum dots captured on the test line. Under optimal conditions, this portable fluorescence biosensor displays rapid responses for nitrated ceruloplasmin with the concentration as low as 1 ng/mL. Furthermore, the biosensor was successfully utilized for spiked human plasma sample detection in a wide dynamic range with a detection limit of 8 ng/mL (S/N = 3). The results demonstrate that the quantum dot-based lateral flow test strip is capable of rapid, sensitive, and quantitative detection of nitrated ceruloplasmin and hold a great promise for point-of-care and in field analysis of other protein biomarkers.

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in hybrid organic-inorganic film of chitosan/sol-gel/carbon nanotubes

A hybrid organic-inorganic nanocomposite film of chitosan/sol-gel/multi-walled carbon nanotubes was constructed for the immobilization of horseradish peroxidase (HRP). This film was characterized by scanning electron microscopy. Direct electron transfer (DET) and bioelectrocatalysis of HRP incorporated into the composite film were investigated. The results indicate that the film can provide a favorable microenvironment for HRP to perform DET on the surface of glassy carbon electrodes with a pair of quasi-reversible redox waves and to retain its bioelectrocatalytic activity toward H(2)O(2).

EQCM Immunoassay for Phosphorylated Acetylcholinesterase as a Biomarker for Organophosphate Exposures Based on Selective Zirconia Adsorption and Enzyme-Catalytic Precipitation

A zirconia (ZrO(2)) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (Phospho-AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO(2) film with uniform nanostructures. The resulting ZrO(2) film was utilized to selectively capture Phospho-AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated proteins. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulation of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus, achieved. Moreover, 4-chloro-1-naphthol (CN) was studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of Phospho-AChE in human plasma with a detection limit of 0.020 nM. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.

Enzyme-Mimic Activity of Ferric Nano-Core Residing in Ferritin and Its Biosensing Applications

Ferritins are nanoscale globular protein cages encapsulating a ferric core. They widely exist in animals, plants, and microbes, playing indispensable roles in iron homeostasis. Interestingly, our study clearly demonstrates that ferritin has an enzyme-mimic activity derived from its ferric nanocore but not the protein cage. Further study revealed that the mimic-enzyme activity of ferritin is more thermally stable and pH-tolerant compared with horseradish peroxidase. Considering the abundance of ferritin in numerous organisms, this finding may indicate a new role of ferritin in antioxidant and detoxification metabolisms. In addition, as a natural protein-caged nanoparticle with an enzyme-mimic activity, ferritin is readily conjugated with biomolecules to construct nanobiosensors, thus holds promising potential for facile and biocompatible labeling for sensitive and robust bioassays in biomedical applications.

Apoferritin nanoparticle: a novel and biocompatible carrier for enzyme immobilization with enhanced activity and stability

Apoferritin is a uniform spherical nano-size biomaterial with excellent bio-compatibility. In this work, we report the use of apoferritin as a novel biocompatible carrier for stabilizing enzymes and enhancing their activities. We used glucose oxidase (GOx) as a model enzyme in this study. GOx was immobilized on the surface of the apoferritin through a green synthetic approach, taking advantage of bioaffinity binding between streptavidin and biotin. As a result, a glucose oxidase–biotin/streptavidin/biotin–apoferritin conjugate (Apo–GOx) was prepared using streptavidin as the bridge. The synthesized Apo–GOx was characterized by transmission electron microscopy, ultraviolet and fluorescence spectroscopy. The activity and stability of GOx on the surface of the apoferritin were investigated and challenged by different environmental factors, such as the temperature, chemicals and pH, in comparison with the biotinylated GOx (B-GOx). The results demonstrate that the activity of Apo-GOx is significantly enhanced while the thermal and chemical stabilities of Apo-GOx are also greatly improved compared to free B-GOx. For instance, the activity of the Apo-GOx only lost 30% after 2 h incubation at 50 oC in comparison to a 70% loss of free B-GOx. The activity of Apo-GOx remains intact after 30 min incubation in 5 M urea solution while B-GOx lost 80% activity after the same treatment. Furthermore, glucose detection was used as a model application for the enzyme immobilization method developed in this work. The GOx immobilized apoferritin nanoparticles exhibited high sensitivity for glucose detection with a detection limit of 3 nM glucose. This work offers a novel approach for immobilizing enzymes with enhanced stability and activity, thus holds the promising advantage for a number of applications, such as in enzyme catalysis, DNA assays and immunoassays.

Sensitive immunoassays of nitrated fibrinogen in human biofluids

Three new sandwich immunoassays for detection of nitrated biomarker have been established with potential applications in biomedical studies and clinical practice. In this study, nitrated human fibrinogen, a potential oxidative stress biomarker for several pathologies, was chosen as the target. To improve the sensitivity and overcome the interference caused by the complexity of human biofluids, we developed three sandwich strategies using various combinations of primary antibody and secondary antibody. All three strategies demonstrated high sensitivity and selectivity towards nitrated forms of fibrinogen in buffer, but their performances were dramatically reduced when tested with human plasma and serum samples. Systematically optimizations were carried out to investigate the effects of numerous factors, including sampling, coating, blocking, and immunoreactions. Our final optimization results indicate that two of these strategies retain sufficient sensitivity and selectivity for use as assays in human physiological samples. Specifically, detection limits reached the pM level and the linear response ranges were up to nM level with a correlation coefficient>0.99. To our best knowledge, this is the first example of using an electrochemical immunoassay for a nitrated biomarker in a physiological fluid. This novel approach provides a rapid, sensitive, selective, cost efficient and robust bioassay for detection of oxidative stress in pathology and for clinical applications. Moreover, the sandwich strategies developed in this paper can be readily used to establish effective methods targeting other nitration biomarkers.

Functionalization of graphene and graphene oxide for biosensing and imaging

Recent advances in our group about graphene bioapplications are discussed. In particular, the functionalization of graphene and graphene oxide, biosensing and bioimaging by using graphene-based nanomaterials, and some fundamental studies of graphene and graphene oxide have been summarized.

Nanoparticles for enhanced sensitivity in electrochemical immunoassays

In this manuscript, we report on electrochemical biosensors based on various nanoparticles (NPs) as labels for sensitive detection of protein biomarkers. We used silica nanoparticle as a carrier to loading a large amount of electroactive species such as poly(guanine) for sensitive immunoassay of tumor necrosis factor-alpha (TNF-a). We took the advantages of the unique hollow structure and reconstruction properties of apoferritin to prepare Cd3(PO4)2 nanoparticles as labels for sensitive assay of TNF-a. A novel immunochromatographic/electro-chemical biosensor based on quantum dots as labels has also been developed for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. These biosensors are quite sensitive with the detection limit at pM level and these approaches based on nanoparticle labels offer a new avenue for sensitive detection of protein biomarkers.