Shanghao Li

Graphene Oxide as a Quencher for Fluorescent Assay of Amino Acids, Peptides, and Proteins

Understanding the interaction between graphene oxide (GO) and the biomolecules is fundamentally essential, especially for disease- and drug-related peptides and proteins. In this study, GO was found to strongly interact with amino acids (tryptophan and tyrosine), peptides (Alzheimers disease related amyloid beta 1-40 and type 2 diabetes related human islet amyloid polypeptide), and proteins (drug-related bovine and human serum albumin) by fluorescence quenching, indicating GO was a universal quencher for tryptophan or tyrosine related peptides and proteins. The quenching mechanism between GO and tryptophan (Trp) or tyrosine (Tyr) was determined as mainly static quenching, combined with dynamic quenching (Förster resonance energy transfer). Different quenching efficiency between GO and Trp or Tyr at different pHs indicated the importance of electrostatic interaction during quenching. Hydrophobic interaction also participated in quenching, which was proved by the presence of nonionic amphiphilic copolymer Pluronic F127 (PF127) in GO dispersion. The strong hydrophobic interaction between GO and PF127 efficiently blocked the hydrophobic interaction between GO and Trp or Tyr, lowering the quenching efficiency.

Strong and Selective Adsorption of Lysozyme on Graphene Oxide

Biosensing methods and devices using graphene oxide (GO) have recently been explored for detection and quantification of specific biomolecules from body fluid samples, such as saliva, milk, urine, and serum. For a practical diagnostics application, any sensing system must show an absence of nonselective detection of abundant proteins in the fluid matrix. Because lysozyme is an abundant protein in these body fluids (e.g., around 21.4 and 7 μg/mL of lysozyme is found in human milk and saliva from healthy individuals, and more than 15 or even 100 μg/mL in patients suffering from leukemia, renal disease, and sarcoidosis), it may interfere with detections and quantification if it has strong interaction with GO. Therefore, one fundamental question that needs to be addressed before any development of GO based diagnostics method is how GO interacts with lysozyme. In this study, GO has demonstrated a strong interaction with lysozyme. This interaction is so strong that we are able to subsequently eliminate and separate lysozyme from aqueous solution onto the surface of GO. Furthermore, the strong electrostatic interaction also renders the selective adsorption of lysozyme on GO from a mixture of binary and ternary proteins. This selectivity is confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), fluorescence spectroscopy, and UV–vis absorption spectroscopy.

Transferrin conjugated nontoxic carbon dots for doxorubicin delivery to target pediatric brain tumor cells

Among various cancers, pediatric brain tumors represent the most common cancer type in children and the second most common cause of cancer related deaths. Anticancer drugs and therapies, such as doxorubicin (Dox), have severe side effects on patients during chemotherapy, especially for children as their bodies are still under development. These side effects are believed to be due to the lack of a delivery system with high efficacy and targeting selectivity, resulting in serious damages of normal cells. To improve the efficacy and selectivity, the transferrin (Trans) receptor mediated endocytosis can be utilized for drug delivery system design, as transferrin receptors are expressed on the blood brain barrier (BBB) and often over expressed in brain tumor cells. Carbon dots (C-Dots) have recently emerged as benign nanoparticles in biomedical applications owing to their good water solubility, tunable surface functionalities and excellent biocompatibility. The unique characteristics of C-Dots make them promising candidates for drug delivery development. In this study, carbon dots-transferrin-doxorubicin covalent conjugate (C-Dots-Trans-Dox) was synthesized, characterized by different spectroscopic techniques and investigated for the potential application as a drug delivery system for anticancer drug doxorubicin to treat pediatric brain tumors. Our in vitro results demonstrate greater uptake of the C-Dots-Trans-Dox conjugate compared to Dox alone presumably owing to the high levels of transferrin receptors on these tumor cells. Experiment showed that C-Dots-Trans-Dox at 10 nM was significantly more cytotoxic than Dox alone, reducing viability by 14-45%, across multiple pediatric brain tumor cell lines.

Method to Determine Protein Concentration in the Protein-Nanoparticle Conjugates Aqueous Solution Using Circular Dichroism Spectroscopy

Considerable efforts have been made to synthesize and characterize protein-nanoparticle conjugates (protein-NPs) for their promising applications in bionanotechnology. However, protein concentration determination in the protein-NPs has so far not been reported. In this Letter, we present a simple and nondestructive approach to quantify the protein concentration in the protein-NPs aqueous solution using circular dichroism (CD) spectroscopy. Carbon dots (∼4 nm), gold nanoparticles (∼10 nm), and polyethylene glycol (PEG, molecular weight ∼3000) were either physically mixed or covalently conjugated (not in the case of gold nanoparticles) with proteins (human transferrin, human serum albumin, and ovalbumin). We were able to quantify the protein concentration in the protein-nanoparticle conjugates using a calibration curve from the CD spectra.

Aggregation of insulin at the interface.

Insulin has so far been the most important pharmaceutical peptide for diabetes treatment, assisting to regulate carbohydrate and fat metabolism in patients. However, aggregation of insulin occurs readily in almost every biopharmaceutical process, ranging from production, purification, storage, transportation, delivery, to in vivo utilization at the terminal. As interfaces and surfaces are ubiquitous in each process and strongly influence physical/chemical properties of insulin, it is necessary and fundamentally important to investigate the aggregation of insulin at various interfaces, such as aqueous-solid interface, water-oil interface, and air-water interface. The objective of this article is to briefly summarize recent progress on insulin aggregation at different interfaces, with special focus on the air-water interface using the Langmuir monolayer technique.

Interactions between Carbon Nanomaterials and Biomolecules

Interactions between carbon nanomaterials, including carbon dots, fullerene, carbon nanotube, graphene, and graphene oxide, and biomolecules play an important role in the field of nanobiotechnology. Due to the unique properties of carbon nanomaterials and the magnificent features of their colloids, it shows high potential in fibrillation inhibition, high sensitivity sensor fabrication, bioimaging, drug delivery, and other areas. Hereby, we will go over different families of carbon nanomaterials regarding to the interaction between carbon nanomaterials and biomolecules at the interface, and their applications will be reviewed as well.

Interaction between Graphene Oxide and Pluronic F127 at the Air–Water Interface

Triblock copolymer Pluronic F127 (PF127) has previously been demonstrated to disperse graphene oxide (GO) in electrolyte solution and block the hydrophobic interaction between GO and l-tryptophan and l-tyrosine. However, the nature of this interaction between PF127 and GO remains to be characterized and elucidated. In the present study, we aimed to characterize and understand the interaction between GO and PF127 using a 2-dimensional Langmuir monolayer methodology at the air-water interface by surface pressure-area isotherm measurement, stability, adsorption, and atomic force microscopy (AFM) imaging. Based on the observation of surface pressure-area isotherms, adsorption, and stability of PF127 and PF127/GO mixture at the air-water interface, GO is suggested to change the conformation of PF127 at the air-water interface and also drag PF127 from the interface to the bulk subphase. Atomic force microscopy (AFM) image supports this assumption, as GO and PF127 can be observed by spreading the subphase solution outside the compressing barriers, as shown in the TOC graphic.

Crossing the blood-brain-barrier with transferrin conjugated carbon dots: A zebrafish model study.

Drug delivery to the central nervous system (CNS) in biological systems remains a major medical challenge due to the tight junctions between endothelial cells known as the blood-brain-barrier (BBB). Here we use a zebrafish model to explore the possibility of using transferrin-conjugated carbon dots (C-Dots) to ferry compounds across the BBB. C-Dots have previously been reported to inhibit protein fibrillation, and they are also used to deliver drugs for disease treatment. In terms of the potential medical application of C-Dots for the treatment of CNS diseases, one of the most formidable challenges is how to deliver them inside the CNS. To achieve this in this study, human transferrin was covalently conjugated to C-Dots. The conjugates were then injected into the vasculature of zebrafish to examine the possibility of crossing the BBB in vivo via transferrin receptor-mediated endocytosis. The experimental observations suggest that the transferrin-C-Dots can enter the CNS while C-Dots alone cannot.

Enhancing selectivity in spectrofluorimetric determination of tryptophan by using graphene oxide nanosheets.

Reaction of formaldehyde with amino acids followed by oxidation with hydrogen peroxide to produce a fluorophore Norharman product is well known and was used for the spectrofluorimetric determination of l-tryptophan (Trp). This study aimed to use graphene oxide (GO) to enhance the selectivity and sensitivity of Trp in presence of other amino acids and possible interfering compounds. Different parameters such as pH, temperature, incubation time, and concentrations of formaldehyde, H2O2 and GO were studied to optimize the condition of determination. Experimental data showed that the maximum fluorescence intensity was achieved in pH 7.0-9.0 phosphate buffer mixed with 7-10% (v/v) formaldehyde and 1-2% (v/v) H2O2 as oxidizing agent at 60°C for 1h. On the basis of calibration curve of various concentrations of Trp in the presence of 20 μg mL(-1) GO, the lower limit of detection (LOD) of Trp was determined as 0.092 nmol mL(-1) and the lower limit of quantification (LOQ) was 0.3 nmol mL(-1). The selectivity of Trp in presence of other amino acids and possible interfering compounds were studied with and without GO. The data obtained after inner filter effect corrections revealed that the selectivity of Trp in presence of amino acids and other possible interfering agents was improved in the range of 76-96%, compared with that in absence of GO. The enhancement of selectivity in the presence of GO indicates that the Trp and other amino acid and possible interfering compounds were adsorbed by GO, and the selective uptaking of Trp-by the reaction with formaldehyde followed by oxidation with H2O2 at 60°C with high selectivity and sensitivity was achieved successfully.

Determination of the composition, encapsulation efficiency and loading capacity in protein drug delivery systems using circular dichroism spectroscopy.

Peptides and proteins have become very promising drug candidates in recent decades due to their unique properties. However, the application of these drugs has been limited by their high enzymatic susceptibility, low membrane permeability and poor bioavailability when administered orally. Considerable efforts have been made to design and develop drug delivery systems that could transport peptides and proteins to targeted area. Although it is of great importance to determine the composition after loading a drug to the carrier, the ability to do so is significantly limited by current analytical methods. In this letter, five important proteins, α1-antitrypsin, hemoglobin human, human serum albumin, human transferrin and r-globulin were chemically conjugated to two model drug carriers, namely carbon dots and polymer O-(2-carboxyethyl) polyethylene glycol. A simple yet convenient method based on circular dichroism spectroscopy was developed to determine the compositions of the various protein-carrier conjugates.