Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE

Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody-antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.     

Combined enhanced fluorescence and label-free biomolecular detection with a photonic crystal surface

A 2D photonic crystal surface with a different period in each lateral direction is demonstrated to detect biomolecules using two distinct sensing modalities. The sensing mechanisms both rely on the generation of a resonant reflection peak at one of two specific wavelengths, depending on the polarization of light that is incident on the photonic crystal. One polarization results in a resonant reflection peak in the visible spectrum to coincide with the excitation wavelength of a fluorophore, while the orthogonal polarization results in a resonant reflection peak at an infrared wavelength which is used for label-free detection of adsorbed biomolecules. The photonic crystal resonance for fluorescence excitation causes enhanced near fields at the structure surface, resulting in increased signal from fluorophores within 100 nm of the device surface. Label-free detection is performed by illuminating the photonic crystal with white light and monitoring shifts in the peak reflected wavelength of the infrared resonance with a high-resolution imaging detection instrument. Rigorous coupled-wave analysis was used to determine optimal dimensions for the photonic crystal structure, and devices were fabricated using a polymer-based nanoreplica molding approach. Fluorescence-based and label-free detection were demonstrated using arrays of spots of dye-conjugated streptavidin. Quantification of the fluorescent signal showed that the fluorescence output from protein spots on the photonic crystal was increased by up to a factor of 35, and deposited spots were also imaged in the label-free detection mode.     

Electrohydrodynamic‐Induced SERS Immunoassay for Extensive Multiplexed Biomarker Sensing

Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multiple cancer biomarkers considering that single biomarker analysis present inadequate information on the underlying biological transformations. Thus, development of sensitive and selective assays for multiple biomarker detection might improve clinical diagnosis and expedite the treatment process. Herein, a microfluidic platform for the rapid, sensitive, and parallel detection of multiple cancer‐specific protein biomarkers from complex biological samples is presented. This approach utilizes alternating current electrohydrodynamic‐induced surface shear forces that provide exquisite control over fluid flow thereby enhancing target–sensor interactions and minimizing non‐specific binding. Further, the use of surface‐enhanced Raman scattering‐based spectral encoding with individual barcodes for different targets enables specific and simultaneous detection of captured protein biomarkers. Using this approach, the specific and sensitive detection of clinically relevant biomarkers including human epidermal growth factor receptor 2 (HER2); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor; and Mucin 16, cell surface associated (MUC16) at concentrations as low as 10 fg mL^?1 in patient serum is demonstrated. Successful target detection from patient samples further demonstrates the potential of this current approach for the clinical diagnosis, which envisages a clinical translation for a rapid and sensitive appraisal of clinical samples in cancer diagnostics.     

Long-range surface plasmon-enhanced fluorescence spectroscopy biosensor for ultrasensitive detection of E. coli O157:H7

A new biosensor platform for the detection of bacterial pathogens based on long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) is presented. The resonant excitation of LRSP modes provides an enhanced intensity of the electromagnetic field, which is directly translated to an increased strength of fluorescence signal measured upon the capture of target analyte at the sensor surface. LRSPs originate from a coupling of surface plasmons across a thin metallic film embedded in dielectrics with similar refractive indices. With respect to regular surface plasmon-enhanced fluorescence spectroscopy, the excitation of LRSPs offers the advantage of a larger enhancement of the evanescent field intensity and a micrometer probing depth that is comparable to the size of target bacterial pathogens. The potential of the developed sensor platform is demonstrated in an experiment in which the detection of E. coli O157:H7 was carried out using sandwich immunoassays. The limit of detection below 10 cfu mL(-1) and detection time of 40 min were achieved.     

Label-free detection of DNA-binding proteins based on microfluidic solid-state molecular beacon sensor

A solid-state molecular beacon using a gold support as a fluorescence quencher is combined with a polydimethylsiloxane (PDMS) microfluidic channel to construct an optical sensor for detecting single-stranded DNA binding protein (SSBP) and histone protein. The single-stranded DNA-Cy3 probe or double-stranded DNA-Cy3 probe immobilized on the gold surface is prepared for the detection of SSBP or histone, respectively. Due to the different quenching ability of gold to the immobilized single-stranded DNA-Cy3 probe and the immobilized double-stranded DNA-Cy3 probe, low fluorescence intensity of the attached single-stranded DNA-Cy3 is obtained in SSBP detection, whereas high fluorescence intensity of the attached double-stranded DNA-Cy3 is obtained in histone detection. The amounts of SSBP in sample solutions are determined from the degree of fluorescence recovery of the immobilized single-stranded DNA-Cy3 probe, whereas that of histone in sample solutions is determined from the degree of fluorescence quenching of the immobilized double-stranded DNA-Cy3 probe. Using this approach, label-free detection of target proteins at nanomolar concentrations is achieved in a convenient, general, continuous flow format. Our approach has high potential for the highly sensitive label-free detection of various proteins based on binding-induced conformation changes of immobilized DNA probes.     

Apta-biosensors for nonlabeled real time detection of human IgE based on carbon nanotube field effect transistors

In this study, we demonstrated the aptamer-based biosensor (apta-biosensor) using CNT-FET devices for label free detection of allergy diagnosis by IgE detection. In order to detect the IgE, two kinds of receptor (monoclonal IgE antibody and anti-IgE aptamer)-modified CNT-FET devices were fabricated. The binding event of the target IgE onto receptors was detected by monitoring the gating effect caused by the charges of the target proteins. Since the CNT-FET biosensors were used in buffer solution, it was crucial to use small-size receptors like aptamers than whole antibodies so that the charged target IgE could approach the CNT surface within the Debye length distance to give a large gating effect. The results show that CNT-FET biosensors using monoclonal IgE antibody had very low sensitivity (minimum detectable level 1000 ng/mL), while those based on anti-IgE aptamer could detect 50 ng/mL. Moreover, the aptamer-modified CNT-FET herein could successfully block non-target proteins and could selectively detect the target protein in an environment similar to human serum electrolyte. Therefore, aptamer-based CNT-FET devices enable the production of label-free ultrasensitive electronic biosensors to detect clinically important biomarkers for disease diagnosis.     

Colorimetric and ultrasensitive bioassay based on a dual-amplification system using aptamer and DNAzyme

Rapid detection of ultralow amount of biomarkers in a biologically complex mixture remains a major challenge. Herein, we report a novel aptamer-based protein detection assay that integrates two signal amplification processes, namely, polymerase-mediated rolling-circle amplification (RCA) and DNA enzyme-catalyzed colorimetric reaction. The target biomarker is captured in a sandwich assay by primary aptamer-functionalized microbeads (MBs) and a secondary aptamer that is connected to a RCA primer/circular template complex. RCA reaction, which amplifies the single biomarker binding events by a factor of hundreds to thousands (the first amplification) produces a long DNA molecule containing multiple DNAzyme units. The peroxidase-like DNAzyme catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (the second amplification), which generates a blue-green colorimetric signal. This new biosensing platform permits the ultrasensitive, label-free, colorimetric detection of biomarker in real time. Using platelet-derived growth factor B-chain (PDGF-BB) as a model system, we demonstrated that our assay can detect a protein marker specifically in a serum-containing medium, at a concentration as low as 0.2 pg/mL in ～2 h, which rivals traditional assays such as ELISA. We anticipate this simple methodology for biomarker detection can find utility in point-of-care applications.     

Cancer prognostics by direct detection of p53-antibodies on gold surfaces by impedance measurements

The identification and measurement of biomarkers is critical to a broad range of methods that diagnose and monitor many diseases. Serum auto-antibodies are rapidly becoming interesting targets because of their biological and medical relevance. This paper describes a highly sensitive, label-free approach for the detection of p53-antibodies, a prognostic indicator in ovarian cancer as well as a biomarker in the early stages of other cancers. This approach uses impedance measurements on gold microelectrodes to measure antibody concentrations at the picomolar level in undiluted serum samples. The biosensor shows high selectivity as a result of the optimization of the epitopes responsible for the detection of p53-antibodies and was validated by several techniques including microcontact printing, self-assembled-monolayer desorption ionization (SAMDI) mass spectrometry, and adhesion pull-off force by atomic force microscopy (AFM). This transduction method will lead to fast and accurate diagnostic tools for the early detection of cancer and other diseases.     

Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance imaging

A methodology for the detection of protein biomarkers at picomolar concentrations that utilizes surface plasmon resonance imaging (SPRI) measurements of RNA aptamer microarrays is developed. The adsorption of proteins onto the RNA microarray is detected by the formation of a surface aptamer-protein-antibody complex. The SPRI response signal is then amplified using a localized precipitation reaction catalyzed by the enzyme horseradish peroxidase that is conjugated to the antibody. This enzymatically amplified SPRI methodology is first characterized by the detection of human thrombin at a concentration of 500 fM; the appropriate thrombin aptamer for the sandwich assay is identified from a microarray of three potential thrombin aptamer candidates. The SPRI method is then used to detect the protein vascular endothelial growth factor (VEGF) at a biologically relevant concentration of 1 pM. VEGF is a signaling protein that has been used as a serum biomarker for rheumatoid arthritis, breast cancer, lung cancer, and colorectal cancer and is also associated with age-related macular degeneration.     

Cell interaction microarray for blood phenotyping

Microarrays promise great advances in areas of diagnostic testing where there is a need to perform multiple assays in parallel. In the short term, protein microarrays have a greater potential to impact diagnostics than DNA arrays due to their potential for direct sample measurements. Here, we report an antibody microarray technique for selectively recognizing glycan and peptide motifs on the surface of red blood cells. We present results demonstrating the optimization and efficacy of the microarray approach as a highly sensitive and specific microscale multiplex assay for blood typing. We also show that our microarray can be used to screen red blood cell surface antigens using whole blood in a label-free detection mode. Finally, our results indicate this method has potential for broader applications in biochip medicine.     

Plasmon line shaping using nanocrosses for high sensitivity localized surface plasmon resonance sensing

The detection of small changes in the wavelength position of localized surface plasmon resonances in metal nanostructures has been used successfully in applications such as label-free detection of biomarkers. Practical implementations, however, often suffer from the large spectral width of the plasmon resonances induced by large radiative damping in the metal nanocavities. By means of a tailored design and using a reproducible nanofabrication process, high quality planar gold plasmonic nanocavities are fabricated with strongly reduced radiative damping. Moreover, additional substrate etching results in a large enhancement of the sensing volume and a subsequent increase of the sensitivity. Coherent coupling of bright and dark plasmon modes in a nanocross and nanobar is used to generate high quality factor subradiant Fano resonances. Experimental sensitivities for these modes exceeding 1000 nm/RIU with a Figure of Merit reaching 5 are demonstrated in microfluidic ensemble spectroscopy.     

Oriented immobilization of antibodies with GST-fused multiple Fc-specific B-domains on a gold surface

We have constructed a novel platform for the oriented buildup of immunoglobulins on a gold surface for a surface plasmon resonance imaging microarray. To this end, genetically engineered glutathione S-transferase proteins bearing one, two, and three Fc-specific B-domains in protein G from Streptococci (GST-GB1, -GB2, and -GB3, respectively) were produced. In order to tether these GST-GBx proteins specifically, a novel glutathione-derivatized ligand (LA-GSH) was also synthesized from a biaminated tri(ethylene glycol) backbone. Each end of the backbone was further functionalized with a maleimide group for a glutathione modification and a lipoic acid for surface immobilization. The glutathione ligand demonstrated a negligible nonspecific protein adsorption toward other spectator proteins while showing a strong specific association toward GST-GBx proteins. This Fc-specific surface exhibited at least a 2-fold enhancement in the immunoglobulin density (from human and mouse) with its antigen capture capability totally conserved compared to a covalently tethered GBx proteins. A single antibody tethered on the GST-GB3 is estimated to capture two antigens (enhanced green fluorescent protein), and this antigen capture ratio seems to be the most efficient value ever observed.     

Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides

To establish a quantitative, corroborative understanding of observed correlations between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples, we have characterized amine-terminated, single-stranded DNA probes attached to amine-reactive commercial microarray slides and complementary DNA target hybridization using fluorescence imaging, X-ray photoelectron spectroscopy (XPS) and 32P-radiometric assays. Importantly, we have reproduced DNA probe microarray immobilization densities in macroscopic spotted dimensions using high ionic strength, high-concentration DNA probe solutions to permit direct XPS surface analysis of DNA surface chemistry with good reliability and reproducibility. Target capture hybridization efficiency with complementary DNA exhibited an optimum value at intermediate DNA probe immobilization densities. The macroscopic array model provides a new platform for the study of DNA surface chemistry using highly sensitive, quantitative surface analytical techniques (e.g., XPS, ToF-SIMS). Sensitive 32P-DNA radiometric density measurements were calibrated with more routine XPS DNA signals, facilitating future routine DNA density determinations without the use of a hazardous radioactive assay. The objective is to provide new insight into different surface chemistry influences on immobilized DNA probe environments that affect target capture efficiency from solution to improve microarray assay performance.     

Fabrication of carbohydrate microarrays on gold surfaces: direct attachment of nonderivatized oligosaccharides to hydrazide-modified self-assembled monolayers

This paper describes a new and simple microarray platform for presenting multiple nonderivatized oligosaccharides to protein targets, with utility for mapping carbohydrate-protein recognition events. The approach is based on the creation of a hydrazide-derivatized, self-assembled monolayer on a gold surface in a single or two-step procedure, for efficient and selectively oriented anchoring of oligosaccharide probes via their reducing ends, with detection using fluorescence detection of bound proteins. The biggest hurdles in employing gold-based substrate for fluorescence-based microarray detection include fluorescence quenching and nonspecific surface adsorption of proteins. We found that the quenching effect could be minimized by introducing a omega-thiolated fatty acid (C16) self-assembled monolayer between the gold surface and hydrazide groups, followed by detection involving three successive binding protein layers covering the gold surface. In addition, an effective blocking scheme involving poly(ethylene glycol) aldehyde and bovine serum albumin was employed to reduce nonspecific protein adsorption to the chip surface. As proof of principle, we demonstrate here that sulfated oligosaccharide probes from heparin can be effectively and covalently attached without prior derivatization onto the hydrazide-modified, self-assembled monolayer on gold-coated slide surfaces in a microarray format. This platform is used to assess binding of specific heparin-binding protein targets at very high sensitivity, and we also demonstrate that the approach can be extended to nonsulfated sugars. Direct attachment of nonderivatized sugar probes on the chip is advantageous since it avoids the need for laborious prederivatization and cleanup steps. This versatile fluorescence microarray platform provides a facile approach for interrogating multiple carbohydrate-protein interactions in a high-throughput manner and has potential as a common gold surface platform for other diverse interrogations by MALDI-MS, surface plasmon resonance, and quartz crystal microbalances.     

Microarray immunoassay for phenoxybenzoic acid using polymer encapsulated Eu:Gd2O3 nanoparticles as fluorescent labels

Currently, detection in microarray bioanalysis is based mainly on the use of organic dyes. To overcome photobleaching and spectral overlaps we applied a new type of fluorophore, crystalline europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles, as labels in immunoassay microarrays. The Eu:Gd2O3 nanoparticles synthesized by spray pyrolysis offer narrow red emission, large Stokes shift, photostable laser-induced fluorescence with a long lifetime (1 ms). The amino functionalization of the particles was achieved by poly(L-lysine) (PL) encapsulation. The formation of a stable PL shell was confirmed by TEM analysis, colloidal stability studies, and quantification of the surface reactive amino groups. The PL-encapsulated particles were covalently conjugated to antibodies and successfully applied as reporters in a competitive fluorescence microimmunoassay for phenoxybenzoic acid (PBA), a generic biomarker of human exposure to pyrethroid insecticides. Microarrays were fabricated by microcontact printing of BSA-PBA in line patterns (10 x 10 microm). Confocal fluorescence microscopy combined with internal standard (fluorescein) calibration was used for quantitative measurements. The microarray immunoassay demonstrated a limit of detection of 1.4 microg L(-1) PBA. This work suggests the potential application of lanthanide oxide nanoparticles as fluorescent probes in microarray and biosensor technology, immunodiagnostics, and high-throughput screening.     

Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 6-89%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the <2 pg/mL concentration range within a mixed sample.     

Ultrasensitive microarray detection of short RNA sequences with enzymatically modified nanoparticles and surface plasmon resonance imaging measurements

A novel multiplexed method for short RNA detection that employs an enzymatic capture reaction onto DNA-modified silica nanoparticles (SiNPs) followed by nanoparticle-enhanced surface plasmon resonance imaging (SPRI) is demonstrated. SiNPs functionalized with 5'-phosphorylated single stranded DNA (ssDNA) are used with T4 RNA ligase to capture various short 20-24 base single-stranded RNA (ssRNA) oligonucleotides from a target solution. The ssRNA-modified SiNPs are collected from the target solution, specifically adsorbed onto a cDNA microarray and then detected with SPRI. The use of DNA-modified SiNPs to capture ssRNA for profiling has several advantages as compared to a planar SPRI surface bioaffinity adsorption format: (i) the target solution is exposed to a larger total surface area for the RNA ligation reaction; (ii) the SiNPs enhance the diffusion rate of the ssRNA to the surface; (iii) the SiNPs can be collected, washed, and preconcentrated prior to detection; and (iv) the ssRNA-modified SiNPs give an enhanced SPRI signal upon hybridization adsorption to the microarray. Our initial measurements demonstrate that this detection method can be used to detect multiple ssRNA sequences at concentrations as low as 100 fM in 500 μL.     

PECVD coatings for functionalization of point-of-care biosensor surfaces

In early stage disease diagnosis, an accurate and reliable measurement of low concentrations of specific biomarkers is a key need. The detection technique requires the reaction of an antibody, which is generally covalently bound to the biosensor platform, with its antigen. The application of Zeonor^®, a cyclo olefin copolymer (COP) with very low autofluorescence, good optical properties and high precision molding characteristics, as a biosensor platform has been demonstrated recently. Highly reproducible, industrial scale surface chemical modification of the COP plastic for covalent attachment of the biomolecules for specific recognition of the target, together with low non-specific binding of other proteins that may be present in the sample is a key challenge. In this work, the applicability of plasma enhanced chemical vapor deposition (PECVD) process has been demonstrated by depositing varying surface functionalities including amines, carboxylic, mercapto, epoxy and polyethylene glycol functionalities. The plasma functionalized coatings thus created possess both reactive and repellent sites on the biosensor chip, allowing the chip to be configured either for fluorescence or light scattering-based detection or for label-free surface plasmon resonance detection techniques. The versatility of the gas phase deposition process for building sequential chemistries on low cost and disposable plastic chips is presented in detail.     

Development of air-stable, supported membrane arrays with photolithography for study of phosphoinositide-protein interactions using surface plasmon resonance imaging

We report the development of an air-stable, supported membrane array by use of photolithography for label-free detection of lipid-protein interactions. Phosphoinositides and their phosphorylated derivatives (PIPs) were studied for their binding properties to proteins with lipid microarray in combination with surface plasmon resonance imaging (SPRi). We have demonstrated a simple method to fabricate lipid arrays using photoresist and carried out a series of surface characterizations with SPRi, ac impedance, cyclic voltammetry, and fluorescence microscopy to validate the array quality and lipid bilayer formation. A number of lipid compositions have been tested for the robustness of resulting membranes when undergoing dehydration and rehydration procedures, and the 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine/poly(ethylene glycol)-phosphatidylethanolamine (DOPC+/PEG-PE) system stands out as the best performer that yields the recovery to within 2% of the original state according to SPR sensorgrams. Limits of detection on the dehydrated/rehydrated DOPC+/PEG-PE membranes were determined to be 33 nM for avidin binding to biotinylated lipids, 73.5 nM for cholera toxin to GM1, and 25 nM for PtdIns(4,5)P2-binding protein (P(4,5)BP) to PtdIns(4,5)P2 lipid, respectively. These results demonstrate the suitability and sensitivity of this membrane for constructing membrane arrays for SPRi analysis under ambient conditions. With the use of this addressable and functional lipid membrane array, the screening of specific lipid-protein interactions has been conducted. Strong and specific interactions between P(4,5)BP and PtdIns(4,5)P2/DOPC+/PEG-PE membrane were observed as expected, while cross reactions were spotted for P(4,5)BP/PtdIns(4)P and avidin/GM1 at varied degrees. The air-stable membrane array demonstrated here presents a simple, effective approach for constructing functional membrane surfaces for screening applications, which opens a new avenue for the label-free study of membrane proteins and other forms of lipid-membrane interactions.     

Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis

Excessive sample volumes continue to be a major limitation in the analysis of protein-protein interactions, motivating the search for label-free detection methods of greater sensitivity. Herein, we report the first chemical approach for selective protein recognition using fluorescent single-walled carbon nanotubes (SWNTs) enabling label-free microarrays capable of single protein detection. Hexahistidine-tagged capture proteins directly expressed by cell-free synthesis on SWNT/chitosan microarray are bound to a Ni(2+) chelated by Nα,Nα-bis(carboxymethyl)-L-lysine grafted to chitosan surrounding the SWNT. The Ni(2+) acts as a proximity quencher with the Ni(2+)/SWNT distance altered upon docking of analyte proteins. This ability to discern single protein binding events decreases the apparent detection limit from 100 nM, for the ensemble average, to 10 pM for an observation time of 600 s. This first use of cell-free synthesis to functionalize a nanosensor extends this method to a virtually infinite number of capture proteins. To demonstrate this, the SWNT microarrays are used to analyze a network of 1156 protein-protein interactions in the staurosporine-induced apoptosis of SH-SY5Y cells, confirming literature predictions.