Surface-Enhanced Raman Spectroscopy-Based, Homogeneous, Multiplexed Immunoassay with Antibody-Fragments-Decorated Gold Nanoparticles

We report the development of a novel single-step, multiplexed, homogeneous immunoassay platform for sensitive detection of protein targets based on our realization of high surface-enhanced raman spectroscopy (SERS) signal enhancement by controlled assembly of SERS nanoparticles. An essential design of this platform is the use of gold nanoparticles or nanorods codecorated with specially reduced antibody half-fragments, nonfluorescent Raman-active dyes, and passivating proteins as the SERS nanoparticles. These nanoparticles offer a facile approach to accomplish orientational immobilization of antibodies, minimized interparticle distance, multicolor Raman fingerprint coding, low fluorescence background, as well as excellent biocompatibility and stability. Through sandwiched antibody–antigen interactions, controlled assembly of SERS nanoparticles is realized with a strong SERS signal achieved via plasmonic coupling, creating an immunoassay platform for rapid, sensitive, multiplexed quantification of proteins. This platform is demonstrated for reproducible quantification of three cytokines, interferon gamma, interleukin-2, and tumor necrosis factor alpha, with large signal-to-noise ratio. It is also successfully applied to multiplexed cytokine analysis for T cell secretion studies in complicated biological samples. The developed SERS immunoassay platform may create a simple but valuable tool for facilitating accurate validation and early detection of disease biomarkers as well as for point-of-care tests in clinical diagnostics

Enzymatic Immuno-Assembly of Gold Nanoparticles for Visualized Activity Screening of Histone-Modifying Enzymes

Activity screening of histone-modifying enzymes is of paramount importance for epigenetic research as well as clinical diagnostics and therapeutics. A novel biosensing strategy has been developed for sensitive and selective detection of histone-modifying enzymes as well as their inhibitors. This strategy relies on the antibody-mediated assembly of gold nanoparticles (AuNPs) decorated with substrate peptides that are subjected to enzymatic modifications by the histone-modifying enzymes. This design allows a visual and homogeneous assay of the enzyme activity using antibodies without any labels, which circumvents the requirements to prefunctionalize the antibody and affords improved assay simplicity and throughput. Additionally, the use of antibody-based recognition of modified peptides could offer improved specificity as compared with existing techniques based on the enzyme coupled assay. We have demonstrated this strategy using a histone methyltransferase acting on histone H3 (Lys 4) and a histone acetyltransferase acting on histone H3 (Lys 14). The results reveal that the absorption peak characteristic for AuNPs decreases dynamically with increasing activity of the enzymes with concomitant visualizable color attenuation, and subnanomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a robust and convenient visualized platform for screening the enzyme activities and their inhibitors with high sensitivity and selectivity

In Situ Imaging of Individual mRNA Mutation in Single Cells Using Ligation-Mediated Branched Hybridization Chain Reaction (Ligation-bHCR)

Ultrasensitive and specific in situ imaging of gene expression is essential for molecular medicine and clinical theranostics. We develop a novel fluorescence in situ hybridization (FISH) strategy based on a new branched hybridization chain reaction (bHCR) for efficient signal amplification in the FISH assay and a ligase-mediated discrimination for specific mutation detection. To our knowledge, this is the first time that HCR has been realized for mutation detection in the FISH assay. In vitro assay shows that the ligation-bHCR strategy affords high specificity in discriminating single-nucleotide variation in mRNA, and it generates a highly branched polymeric product that confers more efficient amplification or better sensitivity than HCR. Imaging analysis reveals that ligation-bHCR generates highly bright spot-like signals for localization of individual mRNA molecules, and spot signals of different colors are highly specific in genotyping point mutation of individual mRNA. Moreover, this strategy is shown to have the potential for quantitative imaging of the expression of mRNA at the single-cell level. Therefore, this strategy may provide a new promising paradigm in developing highly sensitive and specific FISH methods for various diagnostic and research applications

Enzymatic Control of Plasmonic Coupling and Surface Enhanced Raman Scattering Transduction for Sensitive Detection of DNA Demethylation

We have developed a novel concept for enzymatic control of plasmonic coupling as a surface enhanced Raman scattering (SERS) nanosensor for DNA demethylation. This nanosensor is constructed by decorating gold nanoparticles (AuNPs) with Raman reporters and hemimethylated DNA probes. Demethylation of DNA probes initiates a degradation reaction of the probes by methylation-sensitive endonuclease Bsh 1236I and single-strand selective exonuclease I. This destabilizes AuNPs and mediates the aggregation of AuNPs, generating a strong plasmonic coupling SERS signal in response to DNA demethylation. This nanosensor has the advantages in its high signal-to-noise ratio, superb specificity, and rapid, convenient, and reproducible detection with homogeneous, single-step operation. Thus, it provides a useful platform for detecting DNA demethylation and related molecular diagnostics and drug screening. This work is the first time that enzymatic degradation of DNA substrate probes has been utilized to induce aggregation of AuNPs such that reproducible, sensitive SERS signals can be achieved from biological recognition events. This enzymatic control mechanism for plasmonic coupling may create a new paradigm for the development of SERS nanosensors

Activity-Based DNA-Gold Nanoparticle Probe as Colorimetric Biosensor for DNA Methyltransferase/Glycosylase Assay

We have developed a novel biosensor platform for colorimetric detection of active DNA methyltransferase/glycosylase based on terminal protection of the DNA-gold nanoparticle (AuNP) probes by mechanistically covalent trapping of target enzymes. This biosensor relied on covalent capture of target enzymes by activity-based DNA probes which created terminal protection of the DNA probes tethered on AuNPs from degradation by Exo I and III. This biosensor has the advantages of having highly sensitive, rapid, and convenient detection due to its use of the homogeneous assay format and strong surface plasmon absorption. Because the activity-based probes (ABPs) are mechanistically specific to target enzymes, this strategy also offers improved selectivity and can achieve the information about both abundance and activity of the enzymes. We have demonstrated this strategy using a human DNA (cytosine-5) methyltransferase (Dnmt 1) and a human 8-oxoguanine glycosylase (hOGG 1). The results reveal that the colorimetric response increases dynamically with increasing activity of the enzymes, implying a great potential of this strategy for DNA methyltransferase/glycosylase detection and molecular diagnostics and drug screening. Our strategy can also be used as a promising and convenient approach for visualized screening of ABPs for DNA modifying enzymes

Phospholipid–Graphene Nanoassembly as a Fluorescence Biosensor for Sensitive Detection of Phospholipase D Activity

A novel phospholipid–graphene nanoassembly is developed based on self-assembly of phospholipids on nonoxidative graphene surfaces. The nanoassembly can be prepared easily through noncovalent hydrophobic interactions between the lipid tails and the graphene without destroying the electronic conjugation within the graphene sheet. This imparts the nanoassembly with desired electrical and optical properties with nonoxidative graphene. The phospholipid coating offers excellent biocompatibility, facile solubilization, and controlled surface modification for graphene, making the nanoassembly a useful platform for biofunctionalization of graphene. The nanoassembly is revealed to comprise a bilayer of phospholipids with a reduced graphene oxide sheet hosting in the hydrophobic interior, thus affording a unique planar mimic of the cellular membrane. By using a fluorescein-labeled phospholipid in this nanoassembly, a fluorescence biosensor is developed for activity assay of phospholipase D. The developed biosensor is demonstrated to have high sensitivity, wide dynamic range, and very low detection limit of 0.010 U/L. Moreover, because of its single-step homogeneous assay format it displays excellent robustness, improved assay simplicity and throughput, as well as intrinsic ability to real-time monitor the reaction kinetics

Peptide-Templated Gold Nanocluster Beacon as a Sensitive, Label-Free Sensor for Protein Post-translational Modification Enzymes

Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs’ fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications

Engineering Organelle-Specific Molecular Viscosimeters Using Aggregation-Induced Emission Luminogens for Live Cell Imaging

Subcellular viscosity is essential for cell functions and may indicate its physiological status. We screen two fluorescent probes by engineering tetraphenylethene (TPE) for measuring viscosity in mitochondria and lysosomes, respectively. These two probes are only weakly emissive in nonviscous medium and the emission signals are greatly enhanced in viscous medium due to the restriction of intramolecular motion. The presence of pyridium has endowed one probe with mitochondrial specificity, while the presence of indole ring has granted the other probe with lysosome-targeting ability. Their optical properties are characterized <i>in vitro</i> and their applications in imaging viscosity variations in mitochondria and lysosomes are also demonstrated in living cells under different stimulated processes. In addition, an increase in both mitochondrial and lysosomal viscosity during mitophagy was revealed for the first time with our probes. To our knowledge, this is the first time that TPE is engineered to be fluorescent molecular viscosimeters that possess desirable aqueous solubility, red-shifted emission, and organelle specificity

Endonucleolytic Inhibition Assay of DNA/Fok I Transducer as a Sensitive Platform for Homogeneous Fluorescence Detection of Small Molecule–Protein Interactions

This paper reported a novel homogeneous fluorescence assay strategy for probing small molecule–protein interactions based on endonucleolytic inhibition of a DNA/Fok I transducer. The transducer could cyclically cleave fluorescence-quenched probes to yield activated fluorescence signal, while protein binding to the small molecule label would prevent Fok I from approaching and cleaving the fluorescence-quenched probes. Because of the efficient signal amplification from the cyclic cleavage operation, the developed strategy could offer high sensitivity for detecting small molecule–protein interactions. This strategy was demonstrated using folate and its high-affinity or low-affinity binding proteins. The results revealed that the developed strategy was highly sensitive for detecting either high- or low-affinity small molecule–protein interactions with improved selectivity against nonspecific protein adsorption. This strategy could also be extended for assays of candidate small-molecule ligands using a competitive assay format. Moreover, this strategy only required labeling the small molecule on a DNA heteroduplex, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new method could have potential to become a universal, sensitive, and selective platform for quantitative assays of small molecule–protein interactions

Plasmon Coupling Enhanced Raman Scattering Nanobeacon for Single-Step, Ultrasensitive Detection of Cholera Toxin

We report the development of a novel plasmon coupling enhanced Raman scattering (PCERS) method, PCERS nanobeacon, for ultrasensitive, single-step, homogeneous detection of cholera toxin (CT). This method relies on our design of the plasmonic nanoparticles, which have a bilayer phospholipid coating with embedded Raman indicators and CT-binding ligands of monosialoganglioside (GM1). This design allows a facile synthesis of the plasmonic nanoparticle via two-step self-assembly without any specific modification or chemical immobilization. The realization of tethering GM1 on the surface imparts the plasmonic nanoparticles with high affinity, excellent specificity, and multivalence for interaction with CT. The unique lipid-based bilayer coated structure also affords excellent biocompatibility and stability for the plasmonic nanoparticles. The plasmonic nanoparticles are able to show substantial enhancement of the surface-enhanced Raman scattering (SERS) signals in a single-step interaction with CT, because of their assembly into aggregates in response to the CT-sandwiched interactions. The results reveal that the developed nanobeacon provides a simple but ultrasensitive sensor for rapid detection of CT with a large signal-to-background ratio and excellent reproducibility in a wide dynamic range, implying its potential for point-of-care applications in preventive and diagnostic monitoring of cholera