Biomedical subjects

Xiaodi Su

Publications and source records attributed to Xiaodi Su.

13 recordsLinked to original sources

PubMed2007

Evaluation of two- and three-dimensional streptavidin binding platforms for surface plasmon resonance spectroscopy studies of DNA hybridization and protein-DNA binding.

Surface plasmon resonance (SPR) spectroscopy has been used to study DNA assembly, DNA hybridization, and protein-DNA interactions on two streptavidin (SA) sensor chips. On one chip, SA molecules are immobilized on a biotin-exposed surface, forming an ordered two-dimensional (2D) SA monolayer. The other chip, BIAcore's SA chip, contains SA molecules immobilized within a three-dimensional (3D) carboxylated dextran matrix. Compared to the 2D chip, the 3D SA matrix allows for a slower immobilization rate of biotinylated DNA due to diffusion limitation in the dextran matrix, but with twice the amount of the immobilized DNA due to the greater number of reactive sites, which in turn enables a higher sensitivity for DNA hybridization detection. Interestingly, having a greater DNA probe dispersion in the 3D matrix does not induce a higher DNA hybridization efficiency. In a study of protein binding to immobilized DNA (estrogen receptor to estrogen response elements), aiming at assessing the DNA sequence dependent protein binding behavior, the 2D and 3D chips produce different binding characteristics. On the 2D chip, the protein binding exhibits a better selectivity to the specific sequences, regardless of binding stringency (e.g. salt concentration), whereas on the 3D chip, the liquid handling system needs to be optimized in order to minimize transport limitations and to detect small affinity differences. Through this study we demonstrate that the physicochemical structure of SPR chips affects the apparent binding behaviors of biomolecules. When interpreting SPR binding curves and selecting a sensor chip, these effects should be taken into account.

Biosensing Techniques↗

PubMed2006

Combinational application of surface plasmon resonance spectroscopy and quartz crystal microbalance for studying nuclear hormone receptor-response element interactions.

Conventional methodologies for studying protein-DNA complexes, such as electrophoretic mobility shift assays (EMSAs), lack the real-time sensitivity and precision to accurately characterize the complex dynamics of interactions between transcription factors and their binding sites. To better understand the interactions between estrogen receptor (ER) subtypes and the estrogen response elements (EREs), we employed surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation measurement (QCM-D) and made the following observations: (1) base substitutions in ERE half-sites reduced binding affinity for both ERalpha and ERbeta, (2) ERalpha has a higher sequence specificity than ERbeta or there were more nonspecific interactions between ERbeta and control DNA, and (3) ERalpha bound ERE as dimers and ERbeta bound as tetramers. These findings highlight intrinsic differences in DNA-binding properties between receptor subtypes, which are not apparent based on the high degree of conservation (96% identity) in their DNA-binding domains and results from EMSA studies. With this study, we demonstrate the potential of utilizing SPR and QCM in combination for a comprehensive characterization of ER-DNA interactions, including sequence-dependent binding mechanisms and structural differences in ERalpha-DNA and ERbeta-DNA complexes.

DNA↗

PubMed2006

QCM-D analysis of binding mechanism of phage particles displaying a constrained heptapeptide with specific affinity to SiO2 and TiO2.

A growing number of peptides capable of specifically recognizing inorganic materials have been reported, incrementally increasing the potential to harness peptides as a biological linker to bridge biomolecules and inorganic materials at nanometer scale. In this study, we identified disulfide bond constrained heptapeptides with specific binding affinity to SiO2 and TiO2 using a phage display technique. Interestingly, two of the phage surface displayed peptides enriched with basic amino acid residues, STB1 (HKKPSKS) and STB2 (TKRNNKR), showed a cross binding affinity to both metal oxides. To understand the underlying binding mechanism, binding behaviors of phage particles harboring the STB1 (a high-frequency heptapeptide exhibiting dual binding affinity to both metal oxides) were investigated in a wide pH range using quartz crystal microbalance with energy dissipation measurement (QCM-D). It was found that the binding of STB1-harboring phages to the two metal oxides was clearly mediated by the peptide moiety displayed on the phage surface in a pH-dependent manner, indicating that the binding is largely governed by electrostatic interaction. Furthermore, the interpretation of QCM-D signals (i.e., frequency shift and dissipation shift), with the aid of AFM image analysis of the phage particles bound on the surface of the two metal oxides, elucidated whether the nature of phage (or the displayed peptide) binding to the metal oxides is largely specific or nonspecific.

Amino Acid Sequence↗

PubMed2006

Surface plasmon resonance spectroscopy and electrochemistry study of 4-nitro-1,2-phenylenediamine: a switchable redox polymer with nitro functional groups.

Electrochemistry and electrochemical surface plasmon resonance (SPR) spectroscopy have been applied to study the electrochemical deposition and the redox transition of poly(4-nitro-1,2-phenylenediamine) (P4NoPD) on gold disk. It was shown that SPR can be the signal transducer for the different redox states of P4NoPD. Using a model biomolecular system, involving streptavidin, biotinylated DNA, and its complementary target DNA, it was found that the presence of nitro groups in P4NoPD allows the biorecognition events to be modulated by voltages. There is minimal nonspecific binding of biomolecules on oxidized (+0.2 V) or as-prepared P4NoPD, and binding occurs more significantly on the reduced P4NoPD (-0.2 to -0.6 V) with the presence of amine groups. The electrochemical deposition of P4NoPD film was also conducted on boron-doped diamond (BDD) electrode. The stability of the reduced P4NoPD film on gold and BDD was comparatively evaluated by electrochemical impedance spectroscopy (EIS). The result showed that BDD allows the electrochemical reduction of the P4NoPD film at wider cathodic limits than gold.

Journal Article↗

PubMed2006

Multiplatform genome-wide identification and modeling of functional human estrogen receptor binding sites.

BACKGROUND: Transcription factor binding sites (TFBS) impart specificity to cellular transcriptional responses and have largely been defined by consensus motifs derived from a handful of validated sites. The low specificity of the computational predictions of TFBSs has been attributed to ubiquity of the motifs and the relaxed sequence requirements for binding. We posited that the inadequacy is due to limited input of empirically verified sites, and demonstrated a multiplatform approach to constructing a robust model. RESULTS: Using the TFBS for the estrogen receptor (ER)alpha (estrogen response element [ERE]) as a model system, we extracted EREs from multiple molecular and genomic platforms whose binding to ERalpha has been experimentally confirmed or rejected. In silico analyses revealed significant sequence information flanking the standard binding consensus, discriminating ERE-like sequences that bind ERalpha from those that are nonbinders. We extended the ERE consensus by three bases, bearing a terminal G at the third position 3' and an initiator C at the third position 5', which were further validated using surface plasmon resonance spectroscopy. Our functional human ERE prediction algorithm (h-ERE) outperformed existing predictive algorithms and produced fewer than 5% false negatives upon experimental validation. CONCLUSION: Building upon a larger experimentally validated ERE set, the h-ERE algorithm is able to demarcate better the universe of ERE-like sequences that are potential ER binders. Only 14% of the predicted optimal binding sites were utilized under the experimental conditions employed, pointing to other selective criteria not related to EREs. Other factors, in addition to primary nucleotide sequence, will ultimately determine binding site selection.

Algorithms↗

PubMed2005

Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization.

In this study we evaluate the strengths and weaknesses of surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) technique for studying DNA assembly and hybridization reactions. Specifically, we apply in parallel an SPR instrument and a 5 MHz QCM device with dissipation monitoring (QCM-D) to monitor the assembly of biotinylated DNA (biotin-DNA) on a streptavidin-modified surface and the subsequent target DNA hybridization. Through the parallel measurements, we demonstrate that SPR is more suitable for quantitative analysis of DNA binding amount, which is essential for interfacial DNA probe density control and for the analysis of its effect on hybridization efficiency and kinetics. Although the QCM is not quantitative to the same extent as SPR (QCM measures the total mass of the bound DNA molecules together with the associated water), the dissipation factor of the QCM provides a qualitative measure of the viscoelastic properties of DNA films and the conformation of the bound DNA molecules. The complexity in mass measurement does not impair QCM's potential for a kinetic evaluation of the hybridization processes. For quantification of target DNA, the biotin-DNA modified SPR and QCM sensors are exposed to target DNA with increasing concentration. The plots of SPR/QCM signals versus target DNA concentration show that water entrapment between DNA strands make the QCM sensitivity for the hybridization assay well comparable with that of the SPR, although the intrinsic mass sensitivity of the 5 MHz QCM is approximately 20 times lower.

Biosensing Techniques↗

PubMed2005

Enzyme immobilization on poly(ethylene-co-acrylic acid) films studied by quartz crystal microbalance with dissipation monitoring.

In this study, we use the quartz crystal microbalance with dissipation monitoring (QCM-D) to study the immobilization of the enzyme horseradish peroxidase (HRP) on poly(ethylene-co-acrylic acid) (PEAA) films. The surface polarity of spin-coated PEAA films was varied by heat treatments in air or in a 30% NaOH aqueous solution leading to COOH-depleted or COOH-enriched surfaces, respectively. Two reaction schemes, direct adsorption and amine coupling, were employed for HRP immobilization on the two surfaces. The shifts in frequency and dissipation, Deltaf and DeltaD, measured by QCM-D and the ratio DeltaD/Deltaf were used to evaluate the binding amount and the conformation of the adsorbed enzyme. It is found that HRP immobilized via covalent linkages forms rigid and little dissipative films. In contrast, directly adsorbed HRP films exhibit a highly dissipative structure. HRP-catalyzed oxidation of the 4-chloro-1-naphthol in the presence of H(2)O(2) was used to characterize the catalytic activity of the HRP films. The results show that the enzymatic activity of the covalently immobilized HRP tends to be higher.

Acrylic Resins↗

PubMed2005

Surface plasmon resonance spectroscopy and quartz crystal microbalance study of streptavidin film structure effects on biotinylated DNA assembly and target DNA hybridization.

Surface plasmon resonance (SPR) spectroscopy is employed for the study of biotinylated DNA assembly on streptavidin modified gold surfaces for target DNA hybridization. Two immobilization strategies are involved for constructing streptavidin films, namely, (1) physical adsorption on biotin-containing thiol treated surfaces through biotin-streptavidin links and (2) covalent attachment to 11-mercaptoundecanoic acid (MUA) treated surfaces through amine coupling. To understand the structural properties of the streptavidin films, a quartz crystal microbalance with energy dissipation monitoring (QCM-D) is used to monitor the streptavidin immobilization procedures. The simultaneously measured frequency (Deltaf) and dissipation factor (DeltaD) changes, together with the SPR angle shifts (Deltatheta), suggest that the streptavidin film assembled on the biotin-containing surface is highly rigid with a well-ordered structure while the streptavidin film formed through amine coupling is highly dissipative and less structured. The subsequent biotinylated DNA (biotin-DNA) assembly and target hybridization results show that the streptavidin film structure has distinct effects on the biotin-DNA binding amount. On the streptavidin matrix, not only the probe DNA density but also the strand orientation mediated by the streptavidin films has distinct effects on hybridization efficiency. Particularly, the molecularly ordered streptavidin films formed on the biotin-containing surfaces ensure a well-ordered DNA assembly, which in turn allows for a higher efficiency in target DNA capture and for a higher sensitivity in the hybridization analysis when compared to the biotin-DNA assembled on the less structured streptavidin films formed through amine coupling.

Base Sequence↗

PubMed2005

Surface plasmon resonance spectroscopy and quartz crystal microbalance study of MutS binding with single thymine-guanine mismatched DNA.

MutS is a DNA mismatch binding protein that recognizes heteroduplex DNA containing mispaired or unpaired bases. In this study, we employ a quartz crystal microbalance (QCM) and a surface plasmon resonance (SPR) device for the study of MutS binding with DNA containing a single Thymine-Guanine (T-G) mismatch at different sites. Multi-step surface binding reactions are involved in the study, including probe DNA immobilization on the sensor surface through biotin-streptavidin-biotin bridge chemistry, target DNA hybridization to form T-G heteroduplexes, and MutS recognition of the mutation sites. The QCM frequency (d f) and motional resistance (d R, an impedance parameter reflective of QCM damping), as well as the SPR angle shift (d q ) are recorded for the binding reactions. The combined SPR and QCM data collection and analysis allow for an assessment of not only the amount of bound biopolymer but provide also information on also the structural properties of the streptavidin, DNA and MutS/DNA complexes. The affinity of the MutS/T-G heteroduplex assembly is determined by both the QCM and SPR methods through titration of the surface bound DNA with increasing MutS concentration. It is found that if the T-G mismatch is in the center of the DNA fragment, the MutS/DNA complex is more stable than if the mismatch is located near the end of the fragment.

Base Pair Mismatch↗

PubMed2004

Surface modification studies of edge-oriented molybdenum sulfide nanosheets.

We have synthesized edge-oriented MoS2 nanosheets by the evaporation of a single source precursor based on Mo(IV)-tetrakis(diethylaminodithiocarbomato). The surface chemistry of the MoS2 nanosheets has been studied in order to evaluate the chemical reactivities of the basal planes and edges. By irradiating the MoS2 nanosheet with a scanning infrared laser, micron-scale lithographical structures can be created due to laser-induced oxidation of MoS2 to form nanocrystalline MoO3. Preferential reactivities of the MoS2 basal edges in an electrochemical environment and during vapor phase deposition have been demonstrated. Functionalization of the basal plane with 1-pyrene acetic acid allows the immobilization of DNA and immunoglobins on the MoS2 basal plane.

Journal Article↗

PubMed2004

Detection of point mutation and insertion mutations in DNA using a quartz crystal microbalance and MutS, a mismatch binding protein.

MutS protein is a mismatch binding protein that recognizes mispaired and unpaired base(s) in DNA. In this study, we incorporate the MutS protein-based mutation recognition into quartz crystal microbalance (QCM) measurements for DNA single-base substitution mutation and 1-4 base(s) insertion (or deletion) mutation detection. The method involves the immobilization of single-stranded probe DNA on a QCM surface, the hybridization of target DNA to form homoduplex or heteroduplex DNA, and finally the application of MutS protein for the mutation recognition. By measuring the MutS binding signal, DNA containing a T:G mismatch or unpaired base(s) is(are) discriminated against perfectly matched DNA at target concentrations ranging from 1nM to 5 microM. Furthermore, the QCM damping behavior upon MutS-DNA complex formation is studied using a Network Analyzer. The measured motional resistance changes per coupled MutS unit mass (deltaR/deltaf) are found to be indicative of the viscoelastic or structural properties of the bound protein, corresponding to different binding mechanisms. In addition, the deltaR/deltaf values vary remarkably when the MutS protein binds at different distances away from the QCM surface. Thus, these values can be used as a "fingerprint" for MutS mismatch recognition and also used to quantitatively locate the mutation site.

Adenosine Triphosphatases↗

PubMed2002

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.

Affinity Labels↗

PubMed2002

Quartz tuning fork biosensor.

The use of quartz tuning forks for biosensor applications is investigated. The basis of the sensor is to coat the tuning fork surfaces with specific biomolecules and measure subsequent mass loading from the selective binding of complementary analytes. Two experimental set-ups are evaluated, direct mechanical excitation and self-excitation. Mechanical excitation is achieved by mounting the fork on a piezoelectric plate and it is found that the change in oscillation amplitude on adsorption can be monitored to give the change in mass. However, a major drawback is that the sensitivity is determined by the Q-factor, which varies significantly between different sensors and different experimental arrangements. In self-excitation mode, tuning fork motion is activated and detected by placing the fork within a tuned circuit. Using self-excitation mode, anti-human IgG modified tuning forks can sense the binding of human IgG in the range of 5-100 microg ml(-1). The significance of this study is that quartz tuning forks are routinely made using standard microfabrication process, thus suggesting the possibility of facile microfabrication of arrays of quartz sensors.

Audiometry↗