Epitope-Functionalized Gold Nanoparticles for Rapid and Selective Detection of SARS-CoV-2 IgG Antibodies.

Rapid and inexpensive immunodiagnostic assays to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion are essential for conducting large-scale COVID-19 epidemiological surveillance and profiling humoral responses against SARS-CoV-2 infections or immunizations. Herein, a colorimetic serological assay to detect SARS-CoV-2 IgGs in patients' plasma was developed using short antigenic epitopes conjugated to gold nanoparticles (AuNPs). Four immunodominant linear B-cell epitopes, located on the spike (S) and nucleocapsid (N) proteins of SARS-CoV-2, were characterized for their IgG binding affinity and used as highly specific biological motifs on the nanoparticle to recognize target antibodies. Specific bivalent binding between SARS-CoV-2 antibodies and epitope-functionalized AuNPs trigger nanoparticle aggregation, which manifests as a distinct optical transition in the AuNPs' plasmon characteristics within 30 min of antibody introduction. Co-immobilization of two epitopes improved the assay sensitivity relative to single-epitope AuNPs with a limit of detection of 3.2 nM, commensurate with IgG levels in convalescent COVID-19-infected patients. A passivation strategy was further pursued to preserve the sensing response in human plasma medium. When tested against 35 clinical plasma samples of varying illness severity, the optimized nanosensor assay can successfully identify SARS-CoV-2 infection with 100% specificity and 83% sensitivity. As the epitopes are conserved within the circulating COVID-19 variants, the proposed platform holds great potential to serve as a cost-effective and highly specific alternative to classical immunoassays employing recombinant viral proteins. These epitope-enabled nanosensors further expand the serodiagnostic toolbox for COVID-19 epidemiological study, humoral response monitoring, or vaccine efficiency assessment.

Gold Nanoparticle-Based Förster Resonance Energy Transfer (FRET) Analysis of Estrogen Receptor: DNA Interaction.

Estrogen receptors play critical roles in regulating genes responsible for development and maintenance of reproductive tissues and other physiological function. The interaction of ERs with DNA sequences, known as estrogen response elements (EREs) (a palindromic repeat separated by three-base spacer, 5'GGTCAnnnTGACC-3'), is required for estrogen regulation of target gene expression. Here, we describe a simple "mix-and-measure"-based method for detecting ER:ERE interactions using ERE-immobilized metal nanoparticles and water-soluble conjugated polyelectrolytes (CPEs) as cooperative sensing elements. This method can differentiate the distinct DNA-binding affinity between ERα and ERß, and determine ER:ERE-binding stoichiometry. This method can also accurately detect all 15 singly mutated EREs (i.e., three possible base substitutions at each of one to five positions from left to right of the 5' end half site, GGTCA) for their binding energy to ER. This method is compatible with 96-well plate format for high-throughput analysis.

Fast screening of ligand-protein interactions based on ligand-induced protein stabilization of gold nanoparticles.

High throughput screening of small molecular weight (LMW) ligands for protein and sensitive determination of ligand-induced protein stabilization is an important task in drug discovery and in protein structural and functional genomics studies. In this study, gold nanoparticles (AuNPs) and their aggregation property are used to develop a rapid and less equipment intensive assay for screening the interactions between LMW ligands and transcription factors (TFs) and human serum albumin. The assay is based on the fact that the aggregation/discpersion status of AuNPs is very sensitive to the conformation of surrounding proteins, and when a LMW ligand binds to the proteins, it can enhance proteins' salt and thermal stability, and therefore the protective effect on AuNPs from aggregation. Two TFs, i.e. FoxA1 (Forkhead box A1) and AP-2γ (activating enhancer binding protein 2 gamma), and 14 compounds from an NCI compounds library and human serum albumin (HSA) and three known ligands (ibuprofen, warfarin, and phenytoin) are involved to demonstrate the concept and to prove its generality and robustness. With this AuNP method, two strong LMW binders are identified for FoxA1 and AP-2γ; ligand induced protein stabilization is determined. The results have been verified using surface plasmon resonance spectroscopy (SPR) and differential static light scattering (DSLS) techniques. Tryptophan fluorescent measurement is also conducted to provide further information on protein conformational change upon LMW ligand loading as can be observed from AuNPs' UV-vis spectra. FoxA1 and AP-2γ are pivotal in regulating the transcriptional activity of estrogen receptor alpha and controlling the expression of estrogen-responsive breast cancer cells. Identification of drug candidates targeting these two transcription factors could be an alternative in treating breast cancer, in particular those that have become endocrine resistance.

Studying forkhead box protein A1-DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy.

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods-electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)-as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1-DNA binding. In the AuNPs assay, the distinct ability of protein-DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1-DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.

Hybrid sensor using gold nanoparticles and conjugated polyelectrolytes for studying sequence rule in protein-DNA interactions.

Protein-DNA interactions play center roles in many biological processes. Studying sequence specific protein-DNA interactions and revealing sequence rules require sensitive and quantitative methodologies that are capable of capturing subtle affinity difference with high accuracy and in a high throughput manner. In this study, double stranded DNA-conjugated gold nanoparticles (dsDNA-AuNPs) and water-soluble conjugated polyelectrolytes (CPEs) are used as cooperative sensing elements to construct a suit of hybrid sensors for detecting protein-DNA interactions, exploiting the differential Förster resonance energy transfer (FRET) with and without protein binding. Through a proper selection of CPEs in terms of charge properties relative to the charge of dsDNA-AuNPs and emission wavelengths relative to the AuNP extinction peak, the hybrid sensors can be constructed into "light-on", "light-off", and "two-way" models. Protein binding can be detected by fluorescence recovery, fluorescence quenching, or both ways, respectively. The "two-way" sensor allows for detection of proteins of any charge properties or unknown charge properties. With estrogen receptor (ERα and ERß), their consensus DNA (5'-GGTCAnnnTGACC-5') element, and all 15 possible singly mutated elements (i.e., 3 possible base substitutions at each of 1 to 5 positions from left to right of the 5' end half site, GGTCA), we have demonstrated the accuracy of the hybrids sensors for determination of binding affinity constant, binding stoichiometry, and site- and nucleotide-specific binding energy matrix. The in vitro binding energy determined by the hybrid sensors correlates very well with the energy matrix computed from in vivo genome-wide ERα binding data using Thermodynamic Modeling of ChIP-Seq (rank correlation coefficient 0.98). The high degree of correlation of the in vitro energy matrix versus the in vivo matrix renders the new method a highly reliable alternative for understanding in vivo protein binding in the whole genome.

Study sequence rules of estrogen receptor α-DNA interactions using dual polarization interferometry and computational modeling.

Estrogen receptor α (ERα) is a ligand-activated transcription factor. In a classical model, ERα regulates gene expression by binding to DNA sequences called estrogen response elements (EREs). A perfect ERE contains a palindromic consensus sequence of 5'-GGTCAnnnTGACC-3'. A slight variation in ERE sequence alters ERα binding affinity and, thus, the gene transcription activity. In this study, all possible singly mutated EREs of 15 sequences (three possible base substitutions at each of one to five positions of one half-site) were created. Dual polarization interferometry (DPI) was used to measure the receptor binding to generate an in vitro binding energy model. A motif discovery algorithm, Thermodynamic Modeling of ChIP-seq (TherMos), was used to compute the binding energy model from in vivo genome-wide ERα binding data. The in vitro affinity model measured by DPI correlates very well with the TherMos prediction (in vivo model), with a rank correlation coefficient of 0.91, which indicates that the DPI-determined model is reliable and powerful in understanding of ERα binding in vivo in the whole genome. This is the first report of DPI study of protein-double-stranded DNA (dsDNA) interactions. The assay protocols developed are efficient for screening a large quantity of DNA sequences with single base variation sensitivity.

SPR study of DNA hybridization with DNA and PNA probes under stringent conditions.

Surface plasmon resonance (SPR) spectroscopy has been used for studying on-chip DNA hybridization to a PNA probe and its counterpart DNA probe of a 22-mer sequence. Two stringency control strategies are used for single base mismatch discrimination, namely (1) adding a denaturant, i.e. formamide (FA), into hybridization buffer and (2) coupling negative potentials for selective dehybridization of mismatch DNA. These two strategies have either not been used before or been less-well studied in SPR detection. An end-point SPR measurement protocol (no real-time hybridization profile recorded) is developed for detecting DNA hybridization in the presence of FA, to circumvent the problem that the refractive index of FA is out of the detectable range of the SPR equipment. The missing of real-time measurement of hybridization profile is compensated with QCM measurement. Under optimal conditions, i.e. 10mM PBS with 30% FA and 1mM PBS with 50% FA, single base mismatch DNA is detected with 1.7 and 2.8 times less hybridization signals compared with the perfect match DNA, with the DNA probe and PNA probe, respectively. Under negative potential of -0.2 to -0.4V (vs. Ag/AgCl), mismatch DNA dissociates more than perfect match DNA by 1.7-2.5 times from the DNA probe and 2.1-3.5 times from the PNA probe. The higher mismatch discrimination efficiency of the PNA probe under stringent conditions would be attributable to its higher intrinsic sequence selectivity.

Femtomol SPR detection of DNA-PNA hybridization with the assistance of DNA-guided polyaniline deposition.

The inability of surface plasmon resonance (SPR) spectroscopy to detect extremely small refractive index changes has hindered its applications in ultrasensitive DNA analysis. In this study we report a signal amplification strategy that uses DNA-templated polyaniline deposition, suitable for DNA hybridization analysis with charge neutral peptide nucleic acid (PNA) being probes. Under acidic conditions, protonated aniline monomers are adsorbed on DNA backbones through electrostatic interaction. The microenvironment provided by the DNA facilitates oxidative aniline polymerization initialized by H(2)O(2) in the presence of horseradish peroxide. Under optimal conditions, the detection limit is lowered from 5nM for conventional SPR detection to 0.1pM. The significant sensitivity improvement is attributed to the in-situ polymer chain growth along DNA strands, which introduces drastic refractive index increases. This signal amplification approach does not involve secondary hybridization processes. The detection sensitivity obtained is much better than that of gold nanoparticle-based amplification involving a secondary hybridization process and labeled DNA detection probes.

Bioleaching of spent fluid catalytic cracking catalyst using Aspergillus niger.

The use of the fungus Aspergillus niger for the bioleaching of heavy metals from spent catalyst was investigated, with fluid catalytic cracking (FCC) catalyst as a model. Bioleaching was examined in batch cultures with the spent catalysts at various pulp densities (1-12%). Chemical leaching was also performed using mineral acids (sulphuric and nitric acids) and organic acids (citric, oxalic and gluconic acids), as well as a mixture of organic acids at the same concentrations as that biogenically produced. It was shown that bioleaching realised higher metal extraction than chemical leaching, with A. niger mobilizing Ni (9%), Fe (23%), Al (30%), V (36%) and Sb (64%) at 1% pulp density. Extraction efficiency generally decreased with increased pulp density. Compared with abiotic controls, bioleaching gave rise to higher metal extractions than leaching using fresh medium and cell-free spent medium. pH decreased during bioleaching, but remained relatively constant in both leaching using fresh medium and cell-free spent medium, thus indicating that the fungus played a role in effecting metal extraction from the spent catalyst.