Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2 fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9–mCherry fusion protein was detected 6 h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9–mCherry secretion, demonstrating the utility of this method in a biological experiment.     

Enzymatically biocatalytic precipitates amplified antibody-antigen interaction for super low level immunoassay: an investigation combined surface plasmon resonance with electrochemistry

We demonstrated a simple and efficient strategy, which based on the enzymatically biocatalytic precipitates amplified antibody-antigen interaction, for improving the response signals of surface plasmon resonance (SPR) immunosensing. The antibody-antigen-alkaline phosphatase (AP) labeled secondary antibody sandwich were successfully prepared and characterized by SPR, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The SPR signal amplification was accomplished through probing resonance angle shift and Faradaic electron impedance of [Fe(CN)(6)](3-/4-) redox pair after the enzymatically biocatalytic products precipitating on the immunosensing electrode surface. As a result, the accumulation of the enzymatically biocatalytic precipitates leads to significantly resonance angle shift and increase of electron transfer impedance of [Fe(CN)(6)](3-/4-) probe. The precipitates-enhanced sandwich SPR immunoassay for mouse immunoglobulin G (m-IgG) can easily detect solution protein concentrations in the linear range of 0.02-40 ng mL(-1) and with a detection limit of 200 fg mL(-1), which is more than four-orders and 10 times better compared with the values using streptavidin-biotinylated protein complex and biotinylated HRP biocatalyzation amplification methods. Moreover, this method is generally applicable to other sandwich immunoassays and also can be expanded to monitor other antibody-antigen interaction for immunosensing detection at low concentrations.     

Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical, environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently labeled protein, AlexaFluor647–chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647–chick–MNPs) were used to preconcentrate the target via magnetic separation and as the tracer to demonstrate binding to slides modified with anti-chicken IgG as a capture agent. A full optimization study of the antibody-modified MNPs and their use in the biosensor was performed. This investigation looked at the Alexa647–chick–MNP composition, MNP surface modifications, target preconcentration conditions, and the effect that magnetic extraction has on the Alexa647–chick–MNP binding with the array surface. The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent proteins both for target preconcentration and for subsequent integration into immunoassays performed under flow conditions for enhanced signal generation.     

An indirect competitive immunoassay for insulin autoantibodies based on surface plasmon resonance

We have developed a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D). When measuring trace molecules in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labeled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (4 days to 50min), while sensitivity is maintained at a level comparable to that offered by RIA.     

Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.     

GM‐CSF suppresses antioxidant signaling and drives IL‐1β secretion through NRF2 downregulation

GM‐CSF is a potent inflammatory cytokine regulating myeloid cell differentiation, hematopoiesis, and various other functions. It is functionally associated with a number of inflammatory pathologies including rheumatoid arthritis and inflammatory bowel disease. GM‐CSF has been found to promote NLRP3‐dependent IL‐1β secretion, which may have a significant role in driving inflammatory pathologies. However, the molecular mechanisms remain unknown. Here, we show that GM‐CSF induces IL‐1β secretion through a ROS‐dependent pathway. TNF is required for reactive oxygen species (ROS) generation that strikingly does not promote NLRP3 activation, but instead drives ubiquitylation of IL‐1β, promoting its cleavage through basal NRLP3 activity. GM‐CSF regulates this pathway through suppression of antioxidant responses via preventing upregulation of NRF2. Thus, the pro‐inflammatory effect of GM‐CSF on IL‐1β is through suppression of antioxidant responses, which leads to ubiquitylation of IL‐1β and enhanced processing. This study highlights the role of metabolic regulation of inflammatory signaling and reveals a novel mechanism for GM‐CSF to promote inflammation.     

Real-time monitoring of intracellular signal transduction in PC12 cells by two-dimensional surface plasmon resonance imager

Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. The conventional methods used to observe intracellular reactions have not been convenient with several steps such as labeling and washing steps prior to the readout. Consequently, there is a critical need for label-free observation techniques for monitoring intracellular reactions. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via local refractive index change in PC12 cells adhered on a gold sensor slide without any indicator reagent. PC12 cells were stimulated with KCl and phorbol-12-myristate-13-acetate (PMA, a protein kinase C [PKC] activator) at different concentrations in order to induce intracellular PKC translocation. 2D–SPR signal (reflection intensity change) is very consistent with the cellular response normally detected for these stimulants. Our results suggest that complex intracellular reactions could be real-time monitored and characterized by the 2D–SPR imager. It is further expected that signal transmission that was followed by the translocation of signaling proteins could be observed at the single cell level with the high-resolution 2D–SPR imager.     

CCD camera imaging for the chemiluminescent detection of enzymes using new ultrasensitive reagents

We have designed and constructed an inexpensive imaging system based on charge coupled device (CCD) technology and utilized it to demonstrate the sensitivity and rapid detection possible with Lumigen® chemiluminescent reagents. We also report the development of two new chemiluminescent reagents, Lumi-Phos® Plus and Lumigen PS. Lumi-Phos Plus is an ehanced formulation for the rapid detection of alkaline phosphatase on membranes and in solution. It provides excellent images in blotting applications with exposures of under a minute. Lumigen PS represents a new generation of peroxidase detection reagents. The wide dynamic range with excellent linearity, higher signal and lower background than other chemiluminescent reagents make Lumigen PS of unsurpassed value in enzyme-linked immunoassays and nuclic acid probe assays using HRP conjugates.     

A protein detection technique by using surface plasmon resonance (SPR) with rolling circle amplification (RCA) and nanogold-modified tags

Surface plasmon resonance (SPR) can detect molecules bound to a surface by subtle changes in the SPR angle. By immobilizing probes onto the surface and passing analyte solution through the surface, changes in SPR angle indicate the binding between analyte and probes. Detection of analyte from solution can be achieved easily. By using rolling circle amplification (RCA) and nanogold-modified tags, the signals of analyte binding are greatly amplified, and the sensitivity of this technique is significantly improved. Furthermore, this technique has potentials for ultra-sensitive detection and microarray analysis. In this paper, this detection technique is introduced and shown to have great amplification capability. Using 5 nm nanogold with 30 min of RCA development time, this proposed protein detection technique shows over 60 times amplification of the original signal.     

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 H₂O₂ in the presence of horseradish peroxide. Under optimal conditions, the detection limit is lowered from 5 nM for conventional SPR detection to 0.1 pM. 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.     

Rapid and label-free bacteria detection by surface plasmon resonance (SPR) biosensors

Surface Plasmon Resonance (SPR) biosensor technology has been successfully used for the detection of various analytes such as proteins, drugs, DNA, and microorganisms. SPR-based immunosensors that coupled with a specific antigen-antibody reaction, have become a promising tool for the quantification of bacteria as it offers sensitive, specific, rapid, and label-free detection. In this paper, we review the important issues in the development of SPR-based immunoassays for bacteria detection, concentrating on instrumentation, surface functionalization, liquid handling, and surface regeneration. In addition, this review touches on the recent advances in SPR biosensing for sensitivity enhancement.     

Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

Real-time and Label-free Bio-sensing of Molecular Interactions by Surface Plasmon Resonance: A Laboratory Medicine Perspective

Radioactive, chromogenic, fluorescent and other labels have long provided the basis of detection systems for biomolecular interactions including immunoassays and receptor binding studies. However there has been unprecedented growth in a number of powerful label free biosensor technologies over the last decade. While largely at the proof-of-concept stage in terms of clinical applications, the development of more accessible platforms may see surface plasmon resonance (SPR) emerge as one of the most powerful optical detection platforms for the real-time monitoring of biomolecular interactions in a label-free environment.In this review, we provide an overview of SPR principles and current and future capabilities in a diagnostic context, including its application for monitoring a wide range of molecular markers of disease. The advantages and pitfalls of using SPR to study biomolecular interactions are discussed, with particular emphasis on its potential to differentiate subspecies of analytes and the inherent ability for quantitation through calibration-free concentration analysis (CFCA). In addition, recent advances in multiplex applications, high throughput arrays, miniaturisation, and enhancements using noble metal nanoparticles that promise unprecedented sensitivity to the level of single molecule detection, are discussed.In summary, while SPR is not a new technique, technological advances may see SPR quickly emerge as a highly powerful technology, enabling rapid and routine analysis of molecular interactions for a diverse range of targets, including those with clinical applicability. As the technology produces data quickly, in real-time and in a label-free environment, it may well have a significant presence in future developments in lab-on-a-chip technologies including point-of-care devices and personalised medicine.     

Surface plasmon resonance based pesticide assay on a renewable biosensing surface using the reversible concanavalin A monosaccharide interaction

A competitive immunoassay based on surface plasmon resonance (SPR) for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The novelty of the assay is based on the regeneration of the chip surface by the reversible interaction between monosaccharide (D-glucose) and lectin (Concanavalin A). Concanavalin A-2,4-D conjugate was chemically synthesized, purified and used for binding to the SPR chip modified with covalently bound alpha-D-glucose. The interaction between anti-2,4-D antibody and the surface-bound concanavalin A-2,4-D conjugate was monitored by surface plasmon resonance and the response was used for the quantification of 2,4-D. The dynamic range of the calibration curve was between 3 and 100 ng/ml. The demonstrated principle of surface regeneration based on the reversible sugar-lectin interaction may be of more general applicability in immunoassays.     

Quantitation of amyloid beta peptides Aβ(1-38), Aβ(1-40), and Aβ(1-42) in human cerebrospinal fluid by ultra-performance liquid chromatography-tandem mass spectrometry

Critical events in Alzheimer’s disease (AD) involve an imbalance between the production and clearance of amyloid beta (Aβ) peptides from the brain. Current methods for Aβ quantitation rely heavily on immuno-based techniques. However, these assays require highly specific antibodies and reagents that are time-consuming and expensive to develop. Immuno-based assays are also characterized by poor dynamic ranges, cross-reactivity, matrix interferences, and dilution linearity problems. In particular, noncommercial immunoassays are especially subject to high intra- and interassay variability because they are not subject to more stringent manufacturing controls. Combinations of these factors make immunoassays more labor-intensive and often challenging to validate in support of clinical studies. Here we describe a mixed-mode solid-phase extraction method and an ultra-performance liquid chromatography tandem mass spectrometry (SPE UPLC–MS/MS) assay for the simultaneous quantitation of Aβ_1–38, Aβ_1–40, and Aβ_1–42 from human cerebrospinal fluid (CSF). Negative ion versus positive ion species were compared using their corresponding multiple reaction monitoring (MRM) transitions, and negative ions were approximately 1.6-fold greater in intensity but lacked selectivity in matrix. The positive ion MRM assay was more than sufficient to quantify endogenous Aβ peptides. Aβ standards were prepared in artificial CSF containing 5% rat plasma, and quality control samples were prepared in three pooled CSF sources. Extraction efficiency was greater than 80% for all three peptides, and the coefficient of variation during analysis was less than 15% for all species. Mean basal levels of Aβ species from three CSF pools were 1.64, 2.17, and 1.26 ng/ml for Aβ_1–38; 3.24, 3.63, and 2.55 ng/ml for Aβ_1–40; and 0.50, 0.63, and 0.46 ng/ml for Aβ_1–42.     

Development of a near-infrared fluorescence ELISA method using tyramide signal amplification

In this study, we applied tyramide signal amplification (TSA) to fluorescence enzyme-linked immunosorbent assay (ELISA) employing horseradish peroxidase (HRP) as the detection enzyme. When used with a human epidermal growth factor ELISA kit, the TSA method led to a >100-fold increase in fluorescence signal intensity in comparison to an unamplified method. It also showed wider dynamic range and better sensitivity compared to a conventional method using tetramethylbenzidine as the HRP substrate.     

Two-dimensional surface plasmon resonance imager: An approach to study neuronal differentiation

There is a growing demand for the development of a new bioanalytical technique that is capable of monitoring neuronal differentiation noninvasively, in real time, and without any fluorescent probes. In a previous article, we demonstrated that a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager was very useful to monitor cell response on chemical stimulation in which protein kinase C (PKC) translocation was related. In the current study, we focused on developing a new method for monitoring neuronal differentiation and examined the application of the high-resolution 2D–SPR imager to monitor neuronal differentiation noninvasively and by a label-free format. We successfully monitored the intracellular signal transduction, which was mainly translocation of PKC in PC12 cells by the 2D–SPR imager, and found that the cells treated with a differentiation factor, nerve growth factor (NGF), showed a remarkable enhancement of 2D–SPR response to muscarine, carbachol, and acetylcholine stimulation. The results demonstrated that 2D–SPR sensing is applicable to in situ assessment of neuronal differentiation and to studying the expression state of the specific receptors in the living state.     

An amplified surface plasmon resonance "turn-on" sensor for mercury ion using gold nanoparticles

Inorganic mercury ion (Hg²⁺) has been shown to coordinate to DNA duplexes that feature thymine–thymine (T–T) base pair mismatches. This observation suggests that an Hg²⁺-induced conformational change in a single-stranded DNA molecule can be used to detect aqueous Hg²⁺. Here, we have developed an analytical method using surface plasmon resonance (SPR) to develop a highly selective and sensitive detection technique for Hg²⁺ that takes advantage of T–Hg²⁺–T coordination chemistry. The general concept used in this approach is that the “turn-on” reaction of a hairpin probe via coordination of Hg²⁺ by the T–T base pair results in a substantial increase in the SPR response, followed by specific hybridization with a gold nanoparticle probe to amplify the sensor performance. Meanwhile, the limit of detection is 1 nM, which is lower than other recently developed techniques. A linear correlation is observed between the measured SPR reflectivity and the logarithm of the Hg²⁺ concentration over the concentration range of 5–5000 nM. Additionally, the SPR system provides high selectivity for Hg²⁺ in the presence of other divalent metal ions up to micromolar concentration levels. The proposed approach is also successfully utilized for the determination of Hg²⁺ in water samples.     

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.     

Proteomics analysis of prefractionated human lumbar cerebrospinal fluid

Cerebrospinal fluid (CSF) is produced by the chorioid plexus in the ventricles. It surrounds the brain and bone marrow, and reflects several different disorders of the central nervous system (CNS). Proteomics has been used to analyze CSF in order to discover disease-associated proteins and to elucidate the basic molecular mechanisms that either cause, or result from, CNS disorders. However, some disease-associated proteins are of low-abundance and are difficult to detect. A low total-protein concentration, a high amount of albumin and immunoglobins, and a wide dynamic range (several orders of magnitude) of protein concentration cause several difficulties in the identification of low-abundance CSF proteins. In this study, advantage was taken of the range of different hydrophobic properties of CSF proteins, and a reversed-phase solid-phase extraction (SPE) cartridge was used to prefractionate human lumbar CSF proteins into three separate fractions prior to two-dimensional gel electrophoresis resolution of the proteome. A portion of the high-abundance CSF proteins were removed from two (eluted with 35% and 50% acetonitrile) of the three fractions. Some trace CSF proteins were preferentially enriched in the two fractions, and many proteins were detected in the two-dimensional (2-D) gels of the two fractions. Among the novel proteins identified, sixty-two protein spots that represent forty-two proteins were characterized. Most of the proteins have not been annotated in any previous 2-D map of human CSF, and several have been implicated in CNS diseases. The prefractionation of CSF proteins with SPE, followed by proteomics analysis, provides a new method to explore low-abundance, disease-specific CSF proteins.