Quan Cheng

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification.

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin-strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10(-11) M to 1 × 10(-6) M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.

An enzyme-free surface plasmon resonance biosensing strategy for detection of DNA and small molecule based on nonlinear hybridization chain reaction.

A label-free and enzyme-free surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive and specific detection of target DNA by employing the nonlinear hybridization chain reaction (HCR) amplification. Nonlinear HCR is a hairpin-free system in which double-stranded DNA monomers could dendritically assemble into highly branched nanostructure upon introducing a trigger sequence. The target DNA partly hybridizes with capture probe on the gold sensing chip and the unpaired fragment of target DNA works as a trigger to initiate the nonlinear HCR, forming a chain-branching growth of DNA dendrimer by self-assembly. Real-time amplified SPR response is observed upon the introduction of nonlinear HCR system. The method is capable of detecting target DNA at the concentration down to 0.85 pM in 60min with a dynamic range from 1 pM to 1000 pM, and could discriminate target DNA from mismatched sequences. This biosensing strategy exhibits good reproducibility and precision, and has been successfully applied for detection of target DNA in complex sample matrices. In addition, the nonlinear HCR based SPR biosensing methodology is extended to the detection of adenosine triphosphate (ATP) by aptamer recognition. Thus, the versatile method might become a potential alternative tool for biomolecule detection in medical research and early clinical diagnosis.

Surface Plasmon Resonance: Material and Interface Design for Universal Accessibility

The ability to monitor biomolecular reactions in a real-time and label free manner is nearly synonymous with the surface plasmon resonance (SPR) technique. At its basis, SPR relies on the interaction of an incoming light source with a thin metallic film in close contact with a prism or grating, which, under the correct experimental conditions, allows for the propagative oscillation of the conduction electrons at the surface of the metallic material. The oscillating, electromagnetic fields are extremely sensitive to small changes in the refractive index within the dielectric near the sensing interface, where various binding events and interactions can be detected. Historically, SPR has seen its predominant use within pharmaceutical research, where commercial instrumentation has been employed for drug discovery, antibody characterization, and studies of protein/enzyme inhibition. There have recently been a number of excellent reviews focused on the various applications in which SPR spectroscopy and its deri...

Efficient label-free chemiluminescent immunosensor based on dual functional cupric oxide nanorods as peroxidase mimics

Dual-functional cupric oxide nanorods (CuONRs) as peroxidase mimics are proposed for the development of a flow-through, label-free chemiluminescent (CL) immunosensor. Forming the basis of this cost-efficient, label-free immunoassay, CuONRs, synthesized using a simple hydrothermal method, were deposited onto epoxy-activated standard glass slides, followed by immobilization of biotinylated capture antibodies through a streptavidin bridge. The CuONRs possess excellent catalytic activity, along with high stability as a solid support. Antigens could then be introduced to the sensing system, forming large immunocomplexes that prevent CL substrate access to the surface, thereby reducing the CL signal in a concentration dependent fashion. Using carcinoembryonic antigen (CEA) as a model analyte, the proposed label-free immunosensor was able to rapidly determine CEA with a wide linear range of 0.1-60ngmL-1 and a low detection limit of 0.05ngmL-1. This nanozyme-based immunosensor is simple, sensitive, cost-efficient, and has the potential to be a very promising platform for fast and efficient biosensing applications.

Thermoresponsive Arrays Patterned via Photoclick Chemistry: Smart MALDI Plate for Protein Digest Enrichment, Desalting, and Direct MS Analysis

Sample desalting and concentration are crucial steps before matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis. Current sample pretreatment approaches require tedious fabrication and operation procedures, which are unamenable to high-throughput analysis and also result in sample loss. Here, we report the development of a smart MALDI substrate for on-plate desalting, enrichment, and direct MS analysis of protein digests based on thermoresponsive, hydrophilic/hydrophobic transition of surface-grafted poly(N-isopropylacrylamide) (PNIPAM) microarrays. Superhydrophilic 1-thioglycerol microwells are first constructed on alkyne-silane-functionalized rough indium tin oxide substrates based on two sequential thiol-yne photoclick reactions, whereas the surrounding regions are modified with hydrophobic 1H,1H,2H,2H-perfluorodecanethiol. Surface-initiated atom-transfer radical polymerization is then triggered in microwells to form PNIPAM arrays, which facilitate sample loading and enrichment of protein digests by concentrating large-volume samples into small dots and achieving on-plate desalting through PNIPAM configuration change at elevated temperature. The smart MALDI plate shows high performance for mass spectrometric analysis of cytochrome c and neurotensin in the presence of 1 M urea and 100 mM NaHCO3, as well as improved detection sensitivity and high sequence coverage for α-casein and cytochrome c digests in femtomole range. The work presents a versatile sample pretreatment platform with great potential for proteomic research.

Plasmonic Sensing with 3D Printed Optics

Three-dimensional (3D) printing has undergone an exponential growth in popularity due to its revolutionary and near limitless manufacturing capabilities. Recent trends have seen this technology utilized across a variety of scientific disciplines, including the measurement sciences, but precise fabrication of optical components for high-performance biosensing has not yet been demonstrated. We report here 3D printing of high-quality, custom prisms by stereolithography that enable Kretschmann-configured plasmonic sensing of bacterial toxins. Simple benchtop polishing procedures render a smooth surface that supports propagation of surface plasmon polaritons with a deposited gold layer, which exhibit high bulk refractive index sensitivities and are capable of discriminating trace levels of cholera toxin on a supported lipid membrane interface. Further evidence of the flexibility of this manufacturing technique is demonstrated with printed prisms of varied geometries and in situ monitoring of nanoparticle growth by total internal reflection spectroscopy. This work represents the first example of 3D printed light-guiding sensing platforms and demonstrates the versatility and broad perspective of 3D printing in optical detection.

Silver decahedral nanoparticles empowered SPR imaging-SELEX for high throughput screening of aptamers with real-time assessment

A highly efficient method for aptamer screening with real-time monitoring of the SELEX process was described by silver decahedra nanoparticles (Ag10-NPs) enhanced surface plasmon resonance imaging (SPRI). A microarray chip was developed by immobilization of target protein (Lactoferrin (Lac)) and control proteins (α-lactalbumin (α), β-lactoglobulin (β), casein, and bovine serum albumin (BSA)) on the biochip surface. Ag10-NPs were conjugated with an ssDNA library (lib) (Ag10-NPs-library) that consisted of a central 40 nt randomized sequence and a 20 nt fixed primer sequence. Introduction of the Ag10-NPs-library to the SPRI flow channels drastically increased the sensitivity of SPRI signal for real-time monitoring of SELEX. The work allows rapid screening of potential targets, and yields nine aptamers with high affinity (nanomolar range) for Lac after only six-rounds of selection. The aptamer Lac 13-26 was then further tested by SPRI, and the results demonstrated that the aptamer had the capacity to be ultra-sensitive for specific detection of Lac. The novel SPRI-SELEX method demonstrated here showed many advantages of real-time evaluation, high throughput, and high efficiency.

Plasmonic nanodisc arrays on calcinated titania for multimodal analysis of phosphorylated peptides

A hybrid material of gold nanodiscs on a calcinated titania nanofilm that allows for selective quantitative and qualitative characterization of surface-enriched phosphopeptides has been designed and reported. Fabrication was realized through a combination of layer-by-layer deposition and high temperature calcination for the titania, and hole-mask colloidal lithography for the plasmonic nanostructures. The morphology of the resulting titania material was rigorously characterized, exhibiting substantially decreased surface roughness, which allows for lithographic fabrication of plasmonic nanostructures. Moreover, high specificity in adsorption and enrichment of phosphopeptides was exhibited, which was verified by LSPR shifts and matching peaks under mass spectrometric analysis. The construction of these biochips should inform other combinatorial nanofabrication techniques, in addition to allowing future phosphoproteomic analyses to be performed in a time and resource-efficient manner.

Multiplex immunoassay of chicken cytokines via highly-sensitive chemiluminescent imaging array

Quantitative detection of multiple chicken cytokines is a good evaluation of cell-mediated immunity in chickens after disease infection or vaccination. However, current assay methods for chicken cytokines cannot meet the needs of clinical diagnosis due to unsatisfactory sensitivity and low assay throughput. Herein, a sensitive chemiluminescence (CL) imaging immunosensor array has been developed for high-throughput detection of multiple chicken cytokines. The chicken cytokines immunosensor array was prepared by assembling different cytokine capture antibodies onto a disposable silanized glass chip, where horseradish peroxidase and antibody-conjugated gold nanoparticles were used as multienzymatic amplification probe for CL imaging signal amplification. By using a sandwich assay mode, the amplified CL signals from each sensing array cell were collected for quantitation. Using chicken interleukin-4 and chicken interferon-γ as model cytokines, this novel multiplexed and amplified method demonstrated simultaneous measurement of the two chicken cytokines in the linear ranges of 0.008-0.12 ng/mL and 0.005-0.20 ng/mL, respectively, which yields limits of detection down to 2 pg/mL and 3 pg/mL. The CL imaging array method reported here also demonstrated high specificity, good repeatability, and high stability and accuracy, providing a novel multiplex immunoassay strategy for highly sensitive and high-throughput detection of chicken cytokines and further disease diagnosis in poultry.

Ultrasensitive Detection of Bacterial Protein Toxins on Patterned Microarray via Surface Plasmon Resonance Imaging with Signal Amplification by Conjugate Nanoparticle Clusters

Sensitive detection and monitoring of biological interactions in a high throughput, multiplexed array format has numerous advantages. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy and SPR imaging via the effect of accumulation of conjugated nanoparticles of varying sizes. Bacterial cholera toxin (CT) was chosen for the demonstration of enhanced immunoassay by SPR. After immobilization of CT on a gold surface, specific recognition is achieved by biotinylated anti-CT. The signal is amplified by the attachment of biotinylated 20 nm AuNP via streptavidin bridge, followed by attachment of 5 nm streptavidin-functionalized Fe3O4NP to the AuNP-biotin surface. The continuous surface binding of two differently sized conjugated nanoparticles effectively increases their packing density on surface and significantly improves SPR detection sensitivity, allowing quantitative measurement of CT at very low concentration. The dense packing of conjugated nanoparticles on the surface was confirmed by atomic force microscopy characterization. SPR imaging of the immunoassay for high-throughput analysis utilized an Au-well microarray that attenuated the background resonance interference on the resulting images. A calibration curve of conjugated nanoparticle binding signal amplification for CT detection based on surface coverage has been obtained that shows a correlation in a range from 6.31 × 10-16 to 2.51 × 10-13 mol/cm2 with the limit of detection of 5.01 × 10-16 mol/cm2. The absolute quantity of detection limit using SPR imaging was 0.25 fmol. The versatile nanoparticles and biotin-streptavidin interaction used here should allow adaptation of this enhancement method to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules.