Prostate-specific antigen immunoassays on the BioCD

The BioCD is a spinning-disc interferometric biosensor on which antibodies are immobilized to capture target antigens from biological samples. In this work, BioCDs measured the interferometric response to prostate-specific antigen (PSA). The ideal detection limit for PSA was determined using a BioCD with 12,500 printed target antibody spots with a corresponding number of reference protein spots. Statistical analysis projects the detection limit of PSA as a function of the number of spots included in the average. When approximately 10,000 spot pairs were averaged, the 3σ detection limit was 60 pg/ml in a 2 mg/ml simple protein background. A standard format for BioCD immunoassays uses 96 wells with 32 target spots paired with reference spots. In serum, the detection limit for this format was 1 ng/ml in 3:1 diluted female human serum using a sandwich assay with a nonfluorescent mass tag.     

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.     

Multiplexed analyte and oligonucleotide detection on microarrays using several redox enzymes in conjunction with electrochemical detection

We show that multiple enzyme tags may be used in an immunoassay format or for the detection of sequence-specific DNA on microarrays. The assays may be multiplexed and monitored under separate solution and voltage differences. Thus, the detection method relies on an electrochemical detection format, whereby multiple enzymes can be sensed. In our case we utilize horseradish peroxidase, laccase, and glucose dehydrogenase as enzymes attached to specific antibodies or to streptavidin.     

Two‐step signal amplification for high‐sensitivity detection of biomarkers using gold nanoparticle–based conjugates

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)‐based, two‐step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96‐well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)‐conjugated detection antibodies were labeled on large‐size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high‐sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate‐specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.     

Enzyme-linked immunosorbent assays for the synthetic steroid gestrinone

Gestrinone is a synthetic steroid hormone with anti-estrogenic and anti-progesterone properties. It is used to treat endometriosis, shrink uterine fibroids and reduce menorrhagia; besides, it has been investigated for use as contraceptive. Also, due to its anabolic effects, it has been included in the banned list of performance enhanced drugs in sport. Polyclonal antibodies raised against bovine serum albumin coupled to gestrinone 3-carboxymethyloxime (3OCMO-G) were used to develop two highly sensitive and specific enzyme-linked immunosorbent assays for gestrinone. One of them, based on direct format, shows a detection limit (LD) of 0.09 ± 0.03 ng L⁻¹. The second assay, hapten-protein coating format, can detect until (LD) 0.14 ± 0.05 ng L⁻¹. Both immunoassays were also highly specific, showing negligible or no cross-reactivity to other anabolic steroids. The developed ELISAs detected lower amounts of gestrinone than those determined by the reference chromatographic HPLC/MS/MS methods. The direct format was applied to quantify this steroid in spiked human urine without sample pre-treatment, with recovery values between 76 and 122%.     

Applications of array biosensor for detection of food allergens

Although food is a necessity, compounds within food products can be dangerous and life-threatening for people with food allergies. These allergy-causing compounds, such as proteins from eggs and milk, must be identified on the labels of commercial products. Unintentional contamination of food with these compounds occurs as a result of storage, manufacturing procedures, or cleaning procedures. A sensitive, specific, and rapid method to identify foods containing allergens is required by the food industry. The array biosensor, a rapid detection system, may provide a solution to this need. The array biosensor performs fluorescent immunoassays on the surface of a planar waveguide by first running samples, then fluorescently labeled antibodies, over a surface patterned with capture antibodies. An optical image is captured by a charged-coupled device camera and converted into fluorescence values. Signal intensity and spot location provide information on the compound and its concentration. The array biosensor has been successfully demonstrated for toxin, bacteria, and virus detection at low levels in under 20 min in food, clinical samples, and environmental matrixes. An assay for detection of ovalbumin as an indicator of egg contamination has been developed with limits of detection of 25 pg/mL in buffer and 1.3 ng/mL (13 ng/g) in non-egg pasta extract (buffer:pasta 10:1, v/w).     

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.     

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.     

Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus

Towards developing rapid and portable diagnostics for detecting zoonotic diseases, we have developed microchip-based electrophoretic immunoassays for sensitive and rapid detection of viruses. Two types of microchip-based electrophoretic immunoassays were developed. The initial assay used open channel electrophoresis and laser-induced fluorescence detection with a labeled antibody to detect influenza virus. However, this assay did not have adequate sensitivity to detect viruses at relevant concentrations for diagnostic applications. Hence, a novel assay was developed that allows simultaneous concentration and detection of viruses using a microfluidic chip with an integrated nanoporous membrane. The size-exclusion properties of the in situ polymerized polyacrylamide membrane are exploited to simultaneously concentrate viral particles and separate the virus/fluorescent antibody complex from the unbound antibody. The assay is performed in two simple steps--addition of fluorescently labeled antibodies to the sample, followed by concentration of antibody-virus complexes on a porous membrane. Excess antibodies are removed by electrophoresis through the membrane and the complex is then detected downstream of the membrane. This new assay detected inactivated swine influenza virus at a concentration four times lower than that of the open-channel electrophoresis assay. The total assay time, including device regeneration, is six minutes and requires <50 microl of sample. The filtration effect of the polymer membrane eliminates the need for washing, commonly required with surface-based immunoassays, increasing the speed of the assay. This assay is intended to form the core of a portable device for the diagnosis of high-consequence animal pathogens such as foot-and-mouth disease. The electrophoretic immunoassay format is rapid and simple while providing the necessary sensitivity for diagnosis of the illness state. This would allow the development of a portable, cost-effective, on-site diagnostic system for rapid screening of large populations of livestock, including sheep, pigs, cattle, and potentially birds.     

Monoclonal Antibodies Application in Lateral Flow Immunochromatographic Assays for Drugs of Abuse Detection

Lateral flow assays (lateral flow immunoassays and nucleic acid lateral flow assays) have experienced a great boom in a wide variety of early diagnostic and screening applications. As opposed to conventional examinations (High Performance Liquid Chromatography, Polymerase Chain Reaction, Gas chromatography-Mass Spectrometry, etc.), they obtain the results of a sample’s analysis within a short period. In resource-limited areas, these tests must be simple, reliable, and inexpensive. In this review, we outline the production process of antibodies against drugs of abuse (such as heroin, amphetamine, benzodiazepines, cannabis, etc.), used in lateral flow immunoassays as revelation or detection molecules, with a focus on the components, the principles, the formats, and the mechanisms of reaction of these assays. Further, we report the monoclonal antibody advantages over the polyclonal ones used against drugs of abuse. The perspective on aptamer use for lateral flow assay development was also discussed as a possible alternative to antibodies in view of improving the limit of detection, sensitivity, and specificity of lateral flow assays.     

Analysis of inflammatory biomarkers from tissue biopsies by chip-based immunoaffinity CE

To aid in the biochemical analysis of human skin biopsies, a semiautomatic chip-based CE system has been developed for measuring inflammatory biomarkers in microdissected areas of the biopsy. Following solubilization of the dissected tissue, the desired biomarkers were isolated by immunoaffinity capture using a panel of 12 antibodies, immobilized on a disposable glass fiber disk, within the extraction port of the chip. The captured analytes were labeled with a 635 nm light-emitting laser dye and electroeluted into the separation channel. Electrophoretic separation of all of the analytes was achieved in 2.2 min with quantification of each peak being performed by online LIF detection and integration of each peak area. Comparison of the results obtained from the chip-based system to those obtained using commercially available high-sensitivity immunoassays demonstrated that the chip-based assay provides a fast, accurate procedure for studying the concentrations of inflammatory biomarkers in complex biological materials. The degree of accuracy and precision achieved by the chip-based CE is comparable to conventional immunoassays and the system is capable of analyzing circa six samples per hour. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different biomedical analyses.     

Enzyme shadowing: using antibody-enzyme dually-labeled magnetic particles for fast bacterial detection

Using magnetic particles and immunoseparation for target recovery and detection has been reported to improve the performance and detection limits of traditional analytical methods. For example, magnetic immunocapture can be coupled to detection in a sandwich format using an antibody (Ab) labeled with a reporter molecule or enzyme. In this work we demonstrate that simultaneous incorporation of capture and reporter biocomponents onto the sensing surface is possible and provides assays that are extremely fast and easy to carry out. As a proof of concept, we have produced dually-labeled magnetic particles, simultaneously functionalized with antibody and reporter enzyme. Subsequent capture of Escherichia coli generates a shadowing effect on the particle surface and interferes with the activity of a number of enzyme units. The decrease in signal recorded is proportional to the bacterial concentration and is specific for the target microorganism, with a detection limit of 10(3)-10(4) cell mL(-1) in an assay of about one hour.     

A new procalcitonin optical immunosensor for POCT applications

A new immunosensor for the determination of procalcitonin was developed. A sandwich assay format was implemented on a polymethylmetacrylate optical biochip, opportunely shaped in order to obtain several flow channels and potentially suitable for point of care testing applications. The sandwich format makes use of two new rat monoclonal antibodies. The capture antibody was covalently immobilised on the surface of the plastic chip, and the detection antibody was labelled with DY647 dye. Different combinations of capture and detection antibodies were investigated, and particular attention was devoted in order to avoid the non-specific adsorption. A limit of detection of 0.088 mg L⁻¹ was achieved within the working range of 0.28–50 mg L⁻¹ in buffer samples. The assay was also implemented in human serum, and 0.2 and 0.7–25 mg L⁻¹ were the attained limit of detection and working range, respectively.     

Multiplexed Electrochemical Cancer Diagnostics With Automated Microfluidics

Microfluidic platforms can lead to miniaturisation, increased throughput and reduced reagent consumption, particularly when the processes are automated. Here, a programmable microcontroller is used for automation of a microfluidic platform configured to electrochemically determine the levels of 8 proteins simultaneously in complex liquid samples. The platform system is composed of a programmable Arduino microcontroller that controls inexpensive valve actuators, pump, magnetic stirrer and electronic display. The programmable microcontroller results in repeatable timing for each step in a complex assay protocol, such as sandwich immunoassays. Application of the platform is demonstrated using a multiplexed electrochemical immunoassay based on capture at the electrode surface of magnetic particles labelled with horseradish peroxidase and detection antibody. The multiplexed assay protocol is completed in less than 30 mins and results in detection of eight proteins associated with prostate cancer. The approach presented can be used to automate and simplify high‐throughput screening campaigns, such as detection of multiple biomarkers in patient samples.     

Sensitive and simultaneous detection of different disease markers using multiplexed gold nanorods

A multiplexed bioanalytical assay is produced by incorporating two types of gold nanorods (GNRs). Besides retaining the desirable features of common GNRs LSPR sensors, this sensor is easy to fabricate and requires only a visible–NIR spectrometer for detection. This assay can simultaneously detect different acceptor–ligand pairs by choosing the proper GNRs possessing various LPWs in a wide detection wavelength range and can be developed into a high-throughput detection method. This bioanalytical assay allows easy detection of human serum specimens infected by S. japonicum and tuberculosis (TB) from human serum specimens (human serum/Tris–HCl buffer ratio = 1:10⁴) without the need for sample pretreatment. The technique is very sensitive compared to other standard methods such as indirect hemagglutination assays (IHA) that require a serum concentration ratio of larger than 1:20 and enzyme-linked immunosorbent assays (ELISA) requiring a ratio larger than 1:100. This methodology can be readily extended to other immunoassays to realize wider diagnostic applications.     

Sensitive and high-throughput analyses of purine metabolites by dynamic pH junction multiplexed capillary electrophoresis: a new tool for metabolomic studies.

On-line sample preconcentration by a dynamic pH junction in conjunction with multiplexed capillary electrophoresis (CE) and UV detection represents a sensitive and high-throughput format for future metabolomic research, such as purine analysis. The optimization of purine focusing can be rapidly assessed by systematically altering the sample matrix properties, such as the buffer co-ion, pH and ionic strength using a 96-capillary array format. This method permits focusing of large sample injection volumes, resulting in over a 50-fold improvement in the concentration sensitivity. The limit of detection (S/N = 3) for purine metabolites was less than 8.0 x 10(-8) M under optimum conditions when using UV absorbance. Dynamic pH junction multiplexed CE demonstrated excellent linearity over a hundred-fold concentration range, as well as low inter-capillary precision in terms of normalized migration times and peak areas. The potential for clinically relevant high-throughput analyses of micromolar amounts of purine metabolites in urine was also demonstrated.     

Protein G-liposomal nanovesicles as universal reagents for immunoassays

To improve the antigen-binding activity of liposome-coupled antibodies and to develop universal liposomal nanovesicles for immunoassays, protein G was conjugated to dye-loaded liposomal nanovesicles for the preparation of immunoliposomes. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), a heterobifunctional cross-linker, was used to modify protein G for conjugation to the liposomal nanovesicles. Liposome immunosorbent assays were used to evaluate the binding ability of protein G after sulfo-SMCC modification, to optimize the protein G density on the liposome surface and to determine the amount of IgG binding to the protein G-liposomal nanovesicles. Test strips coated with a narrow zone of antibodies were used to show the successful conjugation. Immunomagnetic beads were used to demonstrate the feasibility of protein G-tagged universal liposomal nanovesicles for immunoassays. Results indicate that the Fc-binding capacity of protein G decreased by only 5.3% after sulfo-SMCC modification. Antibodies were easily conjugated to universal protein G-liposomal nanovesicles in 30 min. The conjugates (protein G-immunoliposomes) were successfully used in immunomagnetic bead assays for the detection of Escherichia coli O157:H7 with a detection limit of approximately 100 CFU/ml. This work demonstrated that protein G-liposomal nanovesicles are a successful universal reagent for easily coupling antibodies in an active orientation on the liposome surface for use in immunoassays.     

Surface plasmon resonance imaging in arrays coupled with mass spectrometry (SUPRA–MS): proof of concept of on-chip characterization of a potential breast cancer marker in human plasma

Protein biomarker discovery and validation are crucial for diagnosis, prognosis, and theranostics of human pathologies; "omics" approaches bring new insights in this field. In particular, the combination of immuno-sensors in array format with mass spectrometry efficiently extends the classical immunoassay format and includes molecular characterization. Here, we coupled surface plasmon resonance imaging (SPRi) with MALDI-TOF mass spectrometry in a hyphenated technique which enables multiplexed quantification of binding by SPRi and molecular characterization of interacting partners by subsequent MS analysis. This adds specificity, because MS enables differentiation of molecules that are difficult to distinguish by use of antibodies, for example truncation variants or protein isoforms. Proof of concept was established for detection, identification, and characterization of a potential breast cancer marker, the LAG3 protein, at ~1?μg?mL(-1), added to human plasma. The analytical performance of this new method, dubbed "SUPRA-MS", was established, particularly its specificity (S/N > 10) and reliability (100?% LAG3 identification with high significant mascot score >87.9). The adjusted format for rapid, collective, and automated on-chip MALDI-MS analysis is robust at the femtomole level and has numerous potential applications in proteomics.     

Microplate-based screening methods for the efficient development of sandwich immunoassays

The selection of suitable antibodies is a critical step in immunoassay development, since the final assay performance is predetermined by this decision to a large extent. Particularly, the screening for matching pairs in sandwich immunoassays is difficult, if both antibodies are derived from one species or when monoclonal antibodies are only available as cell supernatants. Several microplate-based approaches for in situ labeling of detection antibodies were tested, in order to avoid time consuming purification of antibodies for enzyme conjugate synthesis. We investigated labeling with anti-species antibodies and Fab fragments thereof, labeling with protein G and biotinylation of cell supernatants without prior purification. Antibodies against peanut proteins were used as a model and signal-to-blank ratios were used in all cases as a measure of the antibody pair performance. Amongst the investigated approaches, preincubation of the detection antibody with labeled anti-species antibody turned out to be most suitable under our conditions. Diagrams, showing the performance of all possible antibody combinations, were generated with this method and were compared to results obtained with covalently labeled detection antibodies. Finally, a flowchart is presented, suggesting an efficient strategy for the development of highly sensitive sandwich immunoassays.     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.