Biwu Liu

Hydrogen Peroxide Displacing DNA from Nanoceria: Mechanism and Detection of Glucose in Serum

Hydrogen peroxide (H2O2) is a key molecule in biology. As a byproduct of many enzymatic reactions, H2O2 is also a popular biosensor target. Recently, interfacing H2O2 with inorganic nanoparticles has produced a number of nanozymes showing peroxidase or catalase activities. CeO2 nanoparticle (nanoceria) is a classical nanozyme. Herein, a fluorescently labeled DNA is used as a probe, and H2O2 can readily displace adsorbed DNA from nanoceria, resulting in over 20-fold fluorescence enhancement. The displacement mechanism instead of oxidative DNA cleavage is confirmed by denaturing gel electrophoresis and surface group pKa measurement. This system can sensitively detect H2O2 down to 130 nM (4.4 parts-per-billion). When coupled with glucose oxidase, glucose is detected down to 8.9 μM in buffer. Detection in serum is also achieved with results comparable with that from a commercial glucose meter. With an understanding of the ligand role of H2O2, new applications in rational materials design, sensor development, and drug delivery can be further exploited.

Mechanisms of DNA Sensing on Graphene Oxide

Adsorption of a fluorophore-labeled DNA probe by graphene oxide (GO) produces a sensor that gives fluorescence enhancement in the presence of its complementary DNA (cDNA). While many important analytical applications have been demonstrated, it remains unclear how DNA hybridization takes place in the presence of GO, hindering further rational improvement of sensor design. For the first time, we report a set of experimental evidence to reveal a new mechanism involving nonspecific probe displacement followed by hybridization in the solution phase. In addition, we show quantitatively that only a small portion of the added cDNA molecules undergo hybridization while most are adsorbed by GO to play the displacement role. Therefore, it is possible to improve signaling by raising the hybridization efficiency. A key innovation herein is using probes and cDNA with a significant difference in their adsorption energy by GO. This study offers important mechanistic insights into the GO/DNA system. At the same time, it provides simple experimental methods to study the biomolecular reaction dynamics and mechanism on a surface, which may be applied for many other biosensor systems.

Instantaneous attachment of an ultrahigh density of nonthiolated DNA to gold nanoparticles and its applications.

The last 16 years have witnessed the landmark development of polyvalent thiolated DNA-functionalized gold nanoparticles (AuNPs) possessing striking properties within the emerging field of nanobiotechnology. Many novel properties of this hybrid nanomaterial are attributed to the dense DNA shell. However, the question of whether nonthiolated polyvalent DNA-AuNP could be fabricated with a high DNA density and properties similar to those of its thiolated counterpart has not been explored in detail. Herein, we report that by simply tuning the pH of the DNA-AuNP mixture an ultrahigh capacity of nonthiolated DNA can be conjugated to AuNPs in a few minutes, resulting in polyvalent DNA-AuNP conjugates with cooperative melting behavior, a typical property of polyvalent thiolated DNA-functionalized AuNPs. With this method, large AuNPs (e.g., 50 nm) can be functionalized to achieve the colorimetric detection of sub-nanometer DNA. Furthermore, this fast, stable DNA loading was employed to separate AuNPs of different sizes. We propose that a large fraction of the attached DNAs are adsorbed via one or a few terminal bases to afford the high loading capacity and the ability to hybridize with the complementary DNA. This discovery not only offers a time- and cost-effective way to functionalize AuNPs with a high density of nonthiolated DNA but also provides new insights into the fundamental understanding of how DNA strands with different sequences interact with AuNPs.

Attaching DNA to nanoceria: regulating oxidase activity and fluorescence quenching.

Cerium oxide nanoparticles (nanoceria) have recently emerged as a nanozyme with oxidase activity. In this work, we present a few important interfacial properties of nanoceria. First, the surface charge of nanoceria can be controlled not only by adjusting pH but also by adsorption of simple inorganic anions. Adsorption of phosphate and citrate gives negatively charged surface over a broad pH range. Second, nanoceria adsorbs DNA via the DNA phosphate backbone in a sequence-independent manner; DNA adsorption inhibits its oxidase activity. Other anionic polymers display much weaker inhibition effects. Adsorption of simple inorganic phosphate does not have the inhibition effect. Third, nanoceria is a quencher for many fluorophores. These discoveries provide an important understanding for further use of nanoceria in biosensor development, materials science, and nanotechnology.

Intracellular Detection of ATP Using an Aptamer Beacon Covalently Linked to Graphene Oxide Resisting Nonspecific Probe Displacement

Fluorescent aptamer probes physisorbed on graphene oxide (GO) have recently emerged as a useful sensing platform. A signal is generated by analyte-induced probe desorption. To address nonspecific probe displacement and the false positive signal, we herein report a covalently linked aptamer probe for adenosine triphosphate (ATP) detection. A fluorophore and amino dual modified aptamer was linked to the carboxyl group on GO with a coupling efficiency of ∼50%. The linearity, specificity, stability, and regeneration of the covalent sensor were systematically studied and compared to the physisorbed probe. Both sensors have similar sensitivity, but the covalent one is more resistant to nonspecific probe displacement by proteins. The covalent sensor has a dynamic range from 0.125 to 2 mM ATP in buffer at room temperature and is resistance to DNase I. Intracellular ATP imaging was demonstrated using the covalent sensor, which generated a higher fluorescence signal than the physisorbed sensor. After the cells were stimulated with 5 mM Ca(2+) for ATP production, the intracellular signal enhanced by 31.8%. This work highlights the advantages of covalent aptamer sensors using GO as both a quencher and a delivery vehicle for intracellular metabolite detection.

Molecular Imprinting on Inorganic Nanozymes for Hundred-fold Enzyme Specificity

Enzyme-mimicking nanomaterials (nanozymes) are more cost-effective and robust than protein enzymes, but they lack specificity. Herein, molecularly imprinted polymers were grown on Fe3O4 nanozymes with peroxidase-like activity to create substrate binding pockets. Electron microscopy confirmed a shell of nanogel. By imprinting with an adsorbed substrate, moderate specificity was achieved with neutral monomers. Further introducing charged monomers led to nearly 100-fold specificity for the imprinted substrate over the nonimprinted compared to that of bare Fe3O4. Selective substrate binding was further confirmed by isothermal titration calorimetry. The same method was also successfully applied for imprinting on gold nanoparticles (peroxidase mimics) and nanoceria (oxidase mimics). Molecular imprinting furthers the functional enzyme mimicking aspect of nanozymes, and such hybrid materials will find applications in biosensor development, separation, environmental remediation, and drug delivery.

Fluorescent sensors using DNA-functionalized graphene oxide

In the past few years, graphene oxide (GO) has emerged as a unique platform for developing DNA-based biosensors, given the DNA adsorption and fluorescence-quenching properties of GO. Adsorbed DNA probes can be desorbed from the GO surface in the presence of target analytes, producing a fluorescence signal. In addition to this initial design, many other strategies have been reported, including the use of aptamers, molecular beacons, and DNAzymes as probes, label-free detection, utilization of the intrinsic fluorescence of GO, and the application of covalently linked DNA probes. The potential applications of DNA-functionalized GO range from environmental monitoring and cell imaging to biomedical diagnosis. In this review, we first summarize the fundamental surface interactions between DNA and GO and the related fluorescence-quenching mechanism. Following that, the various sensor design strategies are critically compared. Problems that must be overcome before this technology can reach its full potential are described, and a few future directions are also discussed.

Adsorption of DNA Oligonucleotides by Titanium Dioxide Nanoparticles

Titanium dioxide (TiO2) or titania shows great promise in detoxification and drug delivery. To reach its full potential, it is important to interface TiO2 with biomolecules to harness their molecular recognition function. To this end, DNA attachment is an important topic. Previous work has mainly focused on long double-stranded DNA or single nucleotides. For biosensor development and targeted drug delivery, it is more important to use single-stranded oligonucleotides. Herein, the interaction between fluorescently labeled oligonucleotides and TiO2 nanoparticles is reported. The point of zero charge (PZC) of TiO2 is around 6 in water or acetate buffer; therefore, the particles are positively charged at lower pH. However, if in phosphate or citrate buffer, the particles are negatively charged, even at pH ∼2, suggesting strong adsorption of buffer anions. DNA adsorption takes place mainly via the phosphate backbone, although the bases might also have moderate contributions. Peptide nucleic acids (PNAs) with an amide backbone cannot be adsorbed. DNA adsorption is strongly affected by inorganic anions, where phosphate and citrate can strongly inhibit DNA adsorption. DNA adsorption is promoted by adding salt or lowering pH. DNA adsorption is accompanied with fluorescence quenching, and double-stranded DNA showed reduced quenching, allowing for the detection of DNA using TiO2 nanoparticles.

Surface modification of nanozymes

Nanoparticles and proteins are similar in a number of aspects, and using nanoparticles to mimic the catalytic function of enzymes is an interesting yet challenging task. Impressive developments have been made over the past two decades on this front. The term nanozyme was coined to refer to nanoparticlebased enzyme mimics. To date, many different types of nanozymes have been reported to catalyze a broad range of reactions for chemical, analytical, and biomedical applications. Since chemical reactions happen mainly on the surface of nanozymes, an interesting aspect for investigation is surface modification. In this review, we summarize three types of nanozyme materials catalyzing various reactions with a focus on their surface chemistry. For metal oxides, cerium oxide and iron oxide are discussed as they are the most extensively studied. Then, gold nanoparticles and graphene oxide are reviewed to represent metallic and carbon nanomaterials, respectively. Types of modifications include ions, small molecules, and polymers mainly by physisorption, while in a few cases, covalent modifications were also employed. The functional aspect of such modification is to improve catalytic activity, substrate specificity, and stability. Future perspectives of this field are speculated at the end of this review.

Polarity Control for Nonthiolated DNA Adsorption onto Gold Nanoparticles

Gold nanoparticles (AuNPs) functionalized with thiolated DNA have enabled many studies in nanoscience. The strong thiol/gold affinity and the nanoscale curvature of AuNPs allow the attached DNA to adapt an upright conformation favorable for hybridization. Recently, it has been shown that nonthiolated DNA can also be attached via DNA base adsorption. Without a thiol label, both ends of the DNA and even internal bases could be adsorbed, decreasing the specificity of subsequent molecular recognition reactions. In this work, we employed a modular sequence design approach to systematically study the effect of DNA sequence on adsorption polarity. A block of poly adenine (poly-A) could be used to achieve a high density of DNA attachment. When the poly-A block length is short (e.g., below 5-7), the loading was independent of the block length, and the conjugate cannot hybridize to its cDNA effectively, suggesting a random attachment controlled by adsorption kinetics. Increasing the block length leads to reduced capacity but improved hybridization, suggesting that more DNA with the desired conformation was adsorbed due to the thermodynamic effects of poly-A binding. The design can be further improved by including capping sequences rich in T or G. Finally, a more general double-stranded DNA approach was described to be suitable for DNA that cannot satisfy the above-mentioned design requirements.