Reduction of severe bovine serum associated matrix effects on carboxymethylated dextran coated biosensor surfaces

Surface plasmon resonance (SPR) based biosensor technology has been widely used in life science research for many applications. While the advantages of speed, ruggedness, versatility, sensitivity and reproducibility are often quoted, many researchers have experienced severe problem of non-specific binding (NSB) to chip surfaces when performing analysis of biological samples such as bovine serum. Using the direct measurement of the bovine protein leptin, present in bovine serum samples as a model, a unique buffering system has been developed and optimised which was able to significantly reduce the non-specific interactions of bovine serum components with the carboxymethyl dextran chip (CM5) surface on a Biacore SPR system. The developed NSB buffering system comprised of HBS-EP buffer, containing 0.5 M NaCl, 0.005% CM-dextran, pH 9.0. An average NSB reduction (n = 20) of 85.9% and 87.3% was found on an unmodified CM5 surface and a CM5 with bovine leptin immobilised on the chip surface, respectively. A reduction in NSB of up to 94% was observed on both surfaces. The concentration of the constitutive components and pH of the buffer were crucial in achieving this outcome.     

Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay

A surface plasmon resonance immunoassay has been developed to determine human growth hormone (hGH) directly and without pre-treatment in human serum samples. A binding inhibition immunoassay was employed. Antibody concentration, assay buffer and regeneration solution have been optimized in order to reach the best performance and the lower non-specific binding of the matrix components to the sensor surface. The lowest detection limit was 6 ng/mL, with a working range covering the physiological range. Reproducibility of the assay was excellent with both intra-assay and inter-assay relative standard deviations <5%, while a variation of 2.19% was obtained employing different sensor chips. Reutilization of the sensor surface allows its continuous use over 50 measurements with a signal drop <20%. The SPR immunoassay results were validated using enzyme-linked immunosorbent assay (ELISA) showing an excellent correlation (R² = 0.985). A portable and fully automated system (Sensia SL) was employed in this work. This is the first SPR biosensor assay capable of detecting relevant concentrations of a clinical analyte in serum. This study shows the potentials of this device as a diagnostic tool for the detection of multiple clinical analytes.     

Simultaneous Determination of β2-Microglobulin and Ig E Using Real-Time Biospecific Interaction Analysis (BIA)

Abstract

Real-time biospecific interaction analysis (real time BIA) based on Surface Plasmon Resonance (SPR) has been used to measure the concentration of β2-microglobulin (β2-μ) and Ig E, used as a model system, simultaneously in buffer and plasma. The method relies on specific binding of β2-microglobulin and Ig E to a sensor chip surface where the monoclonal antibodies anti-β2-microglobulin and anti-Ig E were covalently immobilized. The primary binding of the analytes to the surface is followed by the injection, in sequence, of secondary antibodies, each one specific for a different epitope of β2-microglobulin and Ig E, respectively. The binding between the antibodies and the analytes is recorded as an increase in the SPR signal expressed in Resonance Units (RU). The SPR signal is directly related to the amount of β2-microglobulin and Ig E bound to the surface, and depends upon the concentration of the analytes in the sample. The analysis was performed in buffer and serum to show any non-specific binding due to serum proteins. The concentration range 0.35-5.55 nM for both analytes is covered, with good precision and close correlation with an independent standard concentration measurement.     

Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging

A new commercial surface plasmon resonance (SPR) imaging analysis system with a novel SPR dip angle scanning principle allows the measurement, without the need for labeling, of the exact SPR dip angle. With this system hundreds of biomolecular interactions can be monitored on microarrays simultaneously and with great precision. The potency of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. The smallest antibody concentration that could be measured in this experimental setup was 0.5 pM.     

Duplexed sandwich immunoassays on a fiber-optic microarray

In this paper, we describe a duplexed imaging optical fiber array-based immunoassay for immunoglobulin A (IgA) and lactoferrin. To fabricate the individually addressable array, microspheres were functionalized with highly specific monoclonal antibodies. The microspheres were loaded in microwells etched into the distal face of an imaging optical fiber bundle. Two microsphere-based sandwich immunoassays were developed to simultaneously detect IgA and lactoferrin, two innate immune system proteins found in human saliva. Individual microspheres could be interrogated for the simultaneous measurement of both proteins. The working concentration range for IgA detection was between 700 pM and 100 nM, while the working concentration range for lactoferrin was between 385 pM and 10 nM. The cross-reactivity between detection antibodies and their non-specific targets was relatively low in comparison to the signal generated by the specific binding with their targets. These results suggest that the degree of multiplexing on this fiber-optic array platform can be increased beyond a duplex.     

Determination of sulfamethazine in milk by biosensor immunoassay

A biosensor based on surface plasmon resonance (SPR) measurement was developed for use in an immunoassay for detection of sulfamethazine (SMZ) in milk. The biospecific surface was a carboxymethyl dextran-modified gold-surface sensor chip to which SMZ was covalently bound. The assay was based on inhibition of the binding of polyclonal antibodies to immobilized SMZ by SMZ in the sample. The SPR response changed inversely in relation to the antibiotic concentration in the sample. Calibration curves were constructed for SMZ in buffer and in milk at a concentration which included the maximum residue limit (0 to 200 micrograms/kg). The analysis time per sample varied from 8 to 30 min. Different flow rates and antibodies were modified alternatively during the study to assess their influence on the performance of the assay. The active antibody concentration was calculated at approximately 1880 and 180 nM for the antibody anti-SMZ 1 and the antibody anti-SMZ 2, respectively. No cross-reactivity of antibodies with other antibiotics was found. Under optimal conditions, the detection limits in milk for SMZ were 8 and 1.7 micrograms/kg, respectively, for antibody 1 and antibody 2, at a flow rate of 20 microL/min.     

Quantitation of intracellular recombinant human superoxide dismutase using surface plasmon resonance

An immunosensor assay for the quantitation of intracellular recombinant human superoxide dismutase (rhSOD) in Escherichia coli cultivations based on detection with surface plasmon resoance (SPR) is described. A monoclonal antibody for rhSOD was immobilized on a SPR dextran gold chip. Bacterial samples were sonicated and centrifugated prior to injection over the antibody chip for SPR detection. The assay time was 7 min and allowed quantitation in the range of 1-64 nM SOD in lysate samples with a precision of 1.1-3.4%. The assay was applied to monitor the concentration of rhSOD during E. coli bioreactor cultivations where the rhSOD production was induced by iso-propyl-b-d-thiogalactoside (IPTG). The assay allowed accurate monitoring of the production of rhSOD where the important phases in the product formation were possible to see. The report also discusses influence from sample preparation, SPR selectivity and sensitivity and quantitation limits. The assay proved to be fast, sensitive and accurate with low background effects from the dextran matrix of the SPR chip.     

Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor

A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL⁻¹), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.     

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 μg/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HCl buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.     

A Novel IMFET Biosensor Strategy for Interleukin-10 Quantification for Early Screening Heart Failure Disease in Saliva

We propose a salivary Interleukin-10 detection strategy as part of an easily integrable Lab-on-Chip and Point-of-Care IMFET for multi-detection Heart Failure (HF) biomarkers. Our developed IMFET showed good linearity between increasing IL-10 concentration and the charge transfer resistance in both standard solutions and real saliva samples with a LOD of 0.02 pg mL⁻¹. Moreover, the cross-selectivity study showed that the developed IMFET was highly selective towards IL-10 against other HF biomarkers. The precision of our IMFET was also evaluated, and the difference between the determined IL-10 concentration using the standard addition method and its expected one is<20 %.     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators

Nanomolar quantities of single-stranded DNA products ~100 nucleotides long can be detected in diluted 1% serum by surface plasmon resonance (SPR) and film bulk acoustic resonators (FBARs). We have used a novel FBAR sensor in parallel with SPR and obtained promising results with both the acoustic and the optical device. Oligonucleotides and a repellent lipoamide, Lipa-DEA, were allowed to assemble on the sensor chip surfaces for only 15 min by dispensing. Lipa-DEA surrounds the analyte-binding probes on the surface and effectively reduces the non-specific binding of bovine serum albumin and non-complementary strands. In a highly diluted serum matrix, the non-specific binding is, however, a hindrance, and the background response must be reduced. Nanomolar concentrations of short complementary oligos could be detected in buffer, whereas the response was too low to be measured in serum. DNA strands that are approximately 100 base pairs long at concentrations as low as 1-nM could be detected both in buffer and in 1% serum by both SPR and the FBAR resonator.     

CE-based noncompetitive immunoassay for immunoglobulin G in bovine colostrum products

A CE-based noncompetitive immunoassay for IgG in bovine colostrum products was established. FITC-labeled protein G (FITC-PrG) was tagged through noncovalent bindings to the Fc region of the mouse monoclonal antibovine IgG (Ab). The FITC-PrG, Ab, and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. The immunocomplex was separated and analyzed by CZE with LIF detection in less than 2 min in an uncoated fused-silica capillary. Addition of PEG 20,000 (PEG 20M) in the running buffer significantly suppressed analyte adsorption and thus improved the reproducibility and the resolution. The precision of the method was 5.1% (n = 7). A linear relationship was established for the IgG concentration in the range of 1-5 mg/L with a linear correlation coefficient (r = 0.9917). The LOD was 0.1 mg/L (S/N = 3). The method was successfully applied for the determination of IgG in bovine colostrum products and satisfactory results were achieved.     

Direct detection of Δ9-tetrahydrocannabinol in aqueous samples using a homogeneous increasing fluorescence immunoassay (HiFi)

The detection of the major active component of cannabis, Δ9-tetrahydrocannabinol (THC), becomes increasingly relevant due to its widespread abuse. For control purposes, some easy-to-use, sensitive and inexpensive test methods are needed. We have developed a fluorescence immunoassay utilising THC–fluorescein conjugate as tracer. Fluorescence spectroscopy of the conjugate revealed an unusual property: The relatively weak fluorescence of a dilute tracer solution was increased by a factor of up to 5 after binding of a THC-specific antibody. Fluorescence lifetime measurements in aqueous solutions suggested two different tracer conformations both associated with quenching of fluorescein fluorescence by the intramolecular THC moiety. After antibody binding, the tracer enters a third conformation in which fluorescence quenching of fluorescein is completely suppressed. Utilising this property, we established a homogeneous competitive immunoassay (homogeneous increasing fluorescence immunoassay) with low detection limits. The test requires only two reagents, the new tracer molecule and an anti-THC antibody. A single test takes only 8 min. The dynamic detection range for THC is 0.5 to 20 ng/mL in buffer, with a limit of detection (LOD) of 0.5 ng/mL. The test also works in diluted saliva samples (1:10 dilution with buffer) with an LOD of 2 ng/mL and a dynamic range of 2–50 ng/mL.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

Direct surface plasmon resonance immunosensing of pyraclostrobin residues in untreated fruit juices

A surface plasmon resonance (SPR) immunoassay for on-line detection of the strobilurin fungicide pyraclostrobin in untreated fruit juices is presented. The analysis of pyraclostrobin residues is accomplished in apple, grape, and cranberry samples by monitoring the recognition events occurring separately in a two-channel home-made SPR biosensor. Covalent coupling of the analyte derivative results in a reversible method, enabling more than 80 measurements on the same sensor surface. Optimization of the immunoassay conditions provides limits of detection as low as 0.16?μg?L^?1. The selectivity and reproducibility of the analysis is ensured by studying both non-specific interactions with unrelated compounds and inter-assay coefficients of variation. Excellent recovery ranging from 98 to 103?% was achieved by a simple 1:5 dilution of fruit juice with assay buffer before the analysis. The lack of previous cleaning and homogenization procedures reduces the analysis time of a single food sample to only 25?min, including the regeneration cycle.

On-Line Monitoring 1H-Indole-3-Acetic Acid in Plant Tissues Using Molecular Imprinting Monolayer Techniques on a Surface Plasmon Resonance Sensor

A surface plasmon resonance (SPR)-based detection methodology, which allows the identification and quantification of the plant hormone, ¹H-indole-3-acetic acid (IAA) in Peach, Rosa flower, and Crape myrtle bud is described. To achieve high selectivity, a molecularly imprinted monolayer (MIM) was prepared from alkanethiols self-assembled on an SPR sensor chip with preadsorbed template (IAA). The association constant of the MIM to IAA was calculated to be 2.8 × 10¹¹ M^?1 by analysis of the SPR wavenumber changes induced by different concentrations of IAA. A good calibration curve was obtained in a low concentration range of IAA. Moreover, the MIM revealed impressive selectivity with the selectivity factors of 1.0, 0.12, and 0.20 for IAA and other related analogues. Compared to the MIM sensor chip, the affinity of the self-assembled monolayer (SAM) control sensor chip for IAA was significantly low. The single SPR sensor decorated with the MIM was used to determine IAA concentrations in peach, Rosa flower, and Crape myrtle bud, and satisfactory results were obtained. Based on a signal to noise ratio of 3, the detection limits were 0.23 pM for Crape myrtle, 0.20 pM for Peach, and 0.32 pM for Rosa. The method showed good recoveries and precision, 95.7% (RSD 0.54%) for Peach, 98.1% (RSD 3.4%) for Rosa, and 98.1% (RSD 2.5%) for Crape myrtle, spiked with 0.4171 µg, respectively. These results indicate that a combination of SPR sensing and the MIM provides a simple, promising alternative method for determining endogenous IAA in plant tissue in a real-time manner.     

Real-time immunoassay of ferritin using surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used for the first time to determine the concentration of ferritin in both HBS-EP buffer and serum. The monoclonal antibody was immobilized on the carboxymethyl dextran-modified gold surface by an amine coupling method. The interaction of antibody with antigen was monitored in real-time. The signal was enhanced by sandwich amplification strategy to improve the sensitivity and specificity of the immunoassay, especially in serum. The linear range of the assay in serum is over 30-200 ng ml⁻¹ with the detection limit of 28 ng ml⁻¹. The sensitivity, specificity, and reproducibility of the assay are satisfactory. The analyte and enhancement antibody-binding surface could be regenerated by pH 2.0 glycine-HCl buffer and the same antibody-immobilized surface could be used for more than 50 cycles of ferritin binding and regeneration.     

Surface plasmon resonance-based immunoassay for procalcitonin

A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This “single-step” PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4–324 ng mL⁻¹ of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL⁻¹ and 9.2 ng mL⁻¹, respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks.     

Nondestructive monitoring of the photochromic state of dithienylethene monolayers by surface plasmon resonance

The use of surface plasmon resonance (SPR) as a nondestructive, nonerasing readout of the isomerization state of a photochromic dithienylethene covalently linked to a chemically modified gold surface was investigated. Four different binding layers were examined: 11-mercaptoundecanol (MUO), an amine-modified 11-mercaptoundecanol (MUO-NH2), dextran, and an amine-modified dextran. The binding of dithienylethene to the modified gold surface and photoisomerization of the photochrome in the bound state were established by FTIR. Solvent effects were measured for every layer tested using ethanol and hexanes. In general, large, easily measurable SPR signal changes could be detected under conditions where photoisomerization of the dithienylethene photochrome was not quenched by the gold plasmon, establishing SPR as a viable form of readout for potential dithienylethene-based optical data storage or processing devices. Dextran-bound photochrome in ethanol exhibited the largest SPR response upon photoisomerization, but is more prone to time-dependent fluctuations resulting from swelling of the dextran layer (caused by slow diffusion of the solvent) than the other layers. Large responses are also provided by MUO-NH2 and MUO, and the signal is much more stable than that for dextran.