Surface Plasmon Resonance Immunosensor Research Articles

Synthetic peptides-based SPR biosensor evaluation towards canine Visceral leishmaniasis diagnosis: A simple and effective approach

A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor’s analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis, an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol/L); and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs’ samples. For this reason, the PEP13 immunosensor was applied to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.

Interaction Studies of Localized Surface Plasmon Resonance Immunosensor Based on Gold Nanoparticles

Interaction Studies of Localized Surface Plasmon Resonance Immunosensor Based on Gold Nanoparticles

A Novel SPR Immunosensor Based on Dual Signal Amplification Strategy for Detection of SARS-CoV-2 Nucleocapsid Protein

Since the global outbreak of coronavirus disease 2019 (COVID-19), it has spread rapidly around the world. The nucleocapsid (N) protein is one of the most abundant SARS-CoV-2 proteins. Therefore, a sensitive and effective detection method for SARS-CoV-2 N protein is the focus of research. Here, we developed a surface plasmon resonance (SPR) biosensor based on the dual signal-amplification strategy of Au@Ag@Au nanoparticles (NPs) and graphene oxide (GO). Additionally, a sandwich immunoassay was utilized to sensitively and efficiently detect SARS-CoV-2 N protein. On the one hand, Au@Ag@Au NPs have a high refractive index and the capability to electromagnetically couple with the plasma waves propagating on the surface of gold film, which are harnessed for amplifying the SPR response signal. On the other hand, GO, which has the large specific surface area and the abundant oxygen-containing functional groups, could provide unique light absorption bands that can enhance plasmonic coupling to further amplify the SPR response signal. The proposed biosensor could efficiently detect SARS-CoV-2 N protein for 15 min and the detection limit for SARS-CoV-2 N protein was 0.083 ng/mL, with a linear range of 0.1 ng/mL~1000 ng/mL. This novel method can meet the analytical requirements of artificial saliva simulated samples, and the developed biosensor had a good anti-interference capability.

An ultra-sensitive SPR immunosensor for quantitative determination of human cartilage oligomeric matrix protein biomarker

This paper reports the development of a novel surface plasmon resonance (SPR) immunosensor for ultra-sensitive quantitative determination of human articular cartilage oligomeric matrix protein (COMP), a major component of the extracellular matrix and an exploratory biomarker. Capture antibodies against human COMP (anti-COMP16F12) were covalently immobilized on an 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayer (SAM)-coated SPR sensor disk and a dual sandwich-type signal amplification strategy using biotinylated detection antibodies against COMP (anti-COMP17C10-biot) and streptavidin-conjugated quantum dots (SAv‒QDs) were used for the development of an immunosensor. The binding of high-mass SAv‒QDs via biotin–streptavidin interaction to the surface of the immunosensor resulted in a drastic increase in the sensitivity. The developed immunosensor was able to detect concentrations of COMP in a range from 2.80 to 680.54 fM with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.15 and 0.50 fM, respectively. The immunosensor exhibited good repeatability (relative standard deviation (RSD) 8.05%) and reproducibility (RSD 9.88%) as well as excellent operational stability (2.14 % decrease in SPR signal after 13 days). In addition, the analysis of secretomes of human knee articular cartilage explants from patients with osteoarthritis revealed that the immunosensor has good accuracy (analytical error less than 5 %). These results indicate that the immunosensor developed may be suitable for quantitative determination of COMP derived from articular cartilage and other synovial joint tissues in clinical studies.

Trypanosoma evansi RoTat 1.2 variant surface antigen mimotopes selected by panning of the random peptide phage-display library against monoclonal antibodies.

Mimotope peptides of native antigens are valuable for diverse applications such as diagnostics, therapeutics and modern vaccine design. Here, we report for the first time the selection and identification of peptide mimotopes of Trypanosoma evansi RoTat 1.2 variant surface glycoprotein (VSG) for their potential uses in surra diagnostics and multi-epitope vaccine research. First, we produced the mouse monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (IgG1), against the antigens in T. evansi RoTat 1.2 lysates. We then used 2E11 mAb to immunoprecipitate the target antigen. The immunoprecipitated antigen was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in immunoblots. The surface plasmon resonance immunosensors developed with both the mAbs detected VSG in the parasite lysates as well as in the rodent sera. Further, the mAbs were biotinylated and used in three rounds of panning to select peptide mimotopes from the random peptide phage display library (PhD-12; New England Biolabs, USA). The phage clones selected against each mAb were amplified and tested by phage capture ELISA for specificity. The peptide coding regions of the selected phages were sequenced and the protein blast search of the deduced amino acid sequences was performed by accessing the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/. The conformational B epitope prediction of the selected mimotope sequences was done by using 3D Pepitope algorithms accessed at: http://pepitope.tau.ac.il/. The potential applications of the selected mimotopes in surra diagnostics and research are being explored.

Surface Plasmon Resonance Immunosensor with Antibody-Functionalized Magnetoplasmonic Nanoparticles for Ultrasensitive Quantification of the CD5 Biomarker

A surface plasmon resonance (SPR) immunosensor signal amplification strategy based on antibody-functionalized gold-coated magnetic nanoparticles (mAuNPs) was developed for ultrasensitive and quantitative detection of the CD5 biomarker using an indirect sandwich immunoassay format. The gold surface of the SPR sensor disk and mAuNPs was modified with a self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA), and the coupling method using N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide was used to immobilize capture antibodies against human CD5 (anti-CD52A) and detection antibodies against human CD5 (anti-CD52B), respectively. The mAuNPs and anti-CD52B conjugates (mAuNPs–anti-CD52B) were separated by an external magnetic field and used to amplify the SPR signal after the formation of the anti-CD52A/CD5 immune complex on the SPR sensor disk. Compared to the direct CD5 detection with a limit of detection (LOD) of 1.04 nM and a limit of quantification (LOQ) of 3.47 nM, the proposed sandwich immunoassay utilizing mAuNPs–anti-CD52B significantly improved the LOD up to 8.31 fM and the LOQ up to 27.70 fM. In addition, it showed satisfactory performance in human blood serum (recovery of 1.04 pM CD5 was 109.62%). These results suggest that the proposed signal amplification strategy has superior properties and offers the potential to significantly increase the sensitivity of the analysis.

Label-free differentiation and quantification of ricin, abrin from their agglutinin biotoxins by surface plasmon resonance

Here we describe an affinity molecule-directed surface plasmon resonance (SPR) immunosensor for a label-free, differentiation and quantification of ricin and abrin from their structural highly like agglutinin biotoxins. By an introduction of protein G as the affinity capturing molecule, we fulfilled a complete strategy contains (i) screening monoclonal antibodies to be paired in a sandwiched format, (ii) differentiate quantification from the agglutinin, (iii) ascertain of active from inactive biotoxin, and (iv) structural identification of captured biotoxins on a single chip. By the aid of an enrichment step from immunomagnetic beads, we could accurately measure ricin or abrin with a concentration lowered to 0.6 ng/mL (10 pM) in different complex matrices such as stevia, protein powder, and human plasma, with linear ranges of two or three orders of magnitude, and satisfied recovery. We then differentially quantified the mixed crude extracts from castor beans and jequirity peas, and real samples from the fourth OPCW biotoxin exercise to prove the practical availability. We further provided a SPR-mass spectrometric evidence directly obtained from Protein G affinity chip via a noncovalent molecule surface for the first time for definitely structural identification for crude extracts.

Strategy of Dimercaptothiol as Self-assembled Monolayers Enhance the Sensitivity of SPR Immunosensor for Detection of Salbutamol.

The formation of self-assembled monolayers (SAMs) is a normal method for the immobilization of biorecognition elements immobilized on SPR sensors. With this method, mercaptopropionic acid (MPA) with carboxylic and thiol group is the most commonly used. Dimercaptosuccinic acid (DMSA) having two carboxylics and two thiol groups is a classical antidote for heavy metal ions. In this paper, DMSA was first used to form SAMs to connect the antigen on the chip of a surface plasmon resonance (SPR) immunosensor for detection of salbutamol (SAL), and the results were compared with a traditional (MPA)-SPR sensor. Dihydrolipoic acid(DHLA)-SPR showed that the recognition efficiency of antigen and antibody of DMSA-SPR immunosensor was 170.1% at room temperature with the linear range of 5 - 150 ng/mL. The recovery rate of this sensor applied to SAL detection in pork reached 94.9 - 108.0% and the limit of quantification (LOQ) was 5 ng/mL. The results were in good correlation with the analysis results of ultra-high phase liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. This novel DMSA-SPR immunosensor provides insight into a new idea and method for improving the sensitivity of SPR immunosensors and can be widely used in the detection of other small molecules.

Multi-layer optical fiber surface plasmon resonance biosensor based on a sandwich structure of polydopamine-MoSe2@Au nanoparticles-polydopamine.

An all-optical fiber multi-layer surface plasmon resonance (SPR) biosensor based on a sandwich structure of polydopamine-MoSe2@Au nanoparticles-polydopamine (PDA-MoSe2@AuNPs-PDA) was designed for the detection of specific immunoreactions. By optimizing the multi-layer structure and the ratio of MoSe2: AuNPs, a sensitivity of 5117.59 nm/RIU has been obtained, which is more than double that of the only Au-filmed optical fiber SPR sensor. A large surface area was produced by integrating the MoSe2 primitive unit cell and the AuNPs into a hybrid plasmonic nanostructure of MoSe2@AuNPs, leading to optical fiber SPR signal amplification. The nanostructure of MoSe2@AuNPs was surrounded by the PDA layer to guarantee the efficient immobilization of the protein molecules on the optical fiber by strong covalent bond. This biosensor achieved a detection limit of 54.05 ng/mL for detecting the goat-anti-rabbit IgG, which demonstrated enhancements of 12.1%, 23.3% and 184.6% in comparison with three reported SPR biosensors decorated with PDA-AuNPs-PDA, PDA and Cysteamine-MoSe2@AuNPs-Cysteamine nanostructure, respectively. This biosensor achieved favorable selectivity and outstanding sensitivity compared with the reported SPR immuno-sensors, which will provide a miniaturized, rapid-response and label-free optical fiber bio-sensing platform for clinical diagnosis in the future.

Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms.

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.

Simultaneous Detection of Six Different Types of Pesticides by an Immunosensor Based on Surface Plasmon Resonance.

Six pesticides, azoxystrobin, boscalid, chlorfenapyr, imazalil, isoxathion, and nitenpyram, were simultaneously detected by using a surface plasmon resonance (SPR) immunosensor. The working ranges were 3.5 - 19 ng/mL for azoxystrobin, 4.5 - 50 ng/mL for boscalid, 2.5 - 25 ng/mL for chlorfenapyr, 5.5 - 50 ng/mL for imazalil, 3.5 - 50 ng/mL for isoxathion, and 8.5 - 110 ng/mL for nitenpyram. They showed adequate recovery results in tomato samples: 104 - 116% for azoxystrobin, 94 - 101% for boscalid, 90 - 112% for chlorfenapyr, 96 - 106% for imazalil, 107 - 119% for isoxathion, and 104 - 109% for nitenpyram. The correlation coefficient with liquid chromatography (HPLC or LC-MS/MS) using vegetable samples also agreed well: 0.91 - 0.99 as R2 without strong bias, except for nitenpyram for which the SPR immunosensor sensitivity was too low. The SPR immunosensor will have high applicability for pesticide residue analyses in vegetable samples.

Surface plasmon resonance immunosensor for label-free detection of BIRC5 biomarker in spontaneously occurring canine mammary tumours

We report detection of Baculoviral inhibitor of apoptosis repeat containing-5 (BIRC5) protein biomarker in dog serum by label-free surface plasmon resonance (SPR) immunosensor. Initially, overexpression of BIRC5 in canine mammary tumour (CMT) tissues was confirmed by real-time PCR. Recombinant BIRC5 was produced and protein specific antibodies developed in guinea pig specifically reacted with native protein in immunohistochemistry and immunocytochemistry. SPR immunosensor was developed by fabricating anti-BIRC5 antibodies on gold sensor disc. The equilibrium dissociation constant, (KD = kd/ka) was 12.1 × 10−12 M; which indicates that antibodies are of high affinity with sensitivity in picomolar range. The SPR assay could detect as low as 6.25 pg/ml of BIRC5 protein in a calibration experiment (r2 = 0.9964). On testing real clinical samples, 95% specificity and 73.33% sensitivity were recorded. The average amount of serum BIRC5 in dogs with CMT was 110.02 ± 9.77 pg/ml; whereas, in non-cancerous disease conditions, 44.79 ± 4.28 pg/ml and in healthy dog sera 30.28 ± 2.99 pg/ml protein was detected. The SPR immunosensor for detection of BIRC5 in dog sera is reported for the first time and this may find prognostic and diagnostic applications in management of CMT. In future, ‘on-site’ sensors can be developed using this technique for near-patient testing.

Development of a bioaffinity SPR immunosensor based on functionalized graphene oxide for the detection of pregnancy-associated plasma protein A2 in human plasma.

BackgroundGraphene-like material such as functionalized carboxyl-graphene oxide (carboxyl-GO) can be intelligently tuned to achieve particular properties for biological and chemical sensing applications.MethodsIn this study, we propose a method to improve interference of non-specific proteins for use in human plasma assays. The highly specific interactions between molecules are an advantage of carboxyl-GO-based surface plasmon resonance (SPR) immunoassays, and this can be applied to spiked plasma samples with pregnancy-associated plasma protein A2 (PAPPA2).ResultsThe experiment results showed that carboxyl-GO could be used to modulate the plasmon resonance energy, work function and conductivity properties. In addition, carboxyl groups could be used to enhance the conduction of electrons between carboxyl-GO and Au electrodes due to the excellent conductivity and electron transfer rate. The carboxyl-GO-based SPR chip exhibited high sensitivity based on the electric field amplification effects of the composite dielectric material. Therefore, the surface electric field could be enhanced by electron transfer, thereby greatly improving the sensitivity of the sensing system. Enhanced electric field intensity was generated around the carboxyl-GO of 63.58 V/m, and the measured work function was 4.95 eV. The results showed that the carboxyl-GO-based SPR biosensor had high sensitivity, affinity and selective ability for PAPPA2 protein with a high association rate constant (ka) of 3.1 ×109 M-1 S-1 and a limit of detection of 0.01 pg/mL in spiked human plasma.ConclusionThe results showed a detection accuracy of protein in spiked plasma of >90% compared to PBS buffer, suggesting that the carboxyl-GO-based SPR biosensor could be used in assays of human plasma for early and late gynecological diseases. The future of this technology will be useful for the diagnosis and evaluation of the risk of early maternal preeclampsia and potentially in clinical applications for gestational diseases.

Maleic anhydride and acetylene plasma copolymer surfaces for SPR immunosensing

We report on the successful application of carboxyl-rich plasma polymerized (PP) films as a matrix layer for bioreceptor immobilization in surface plasmon resonance (SPR) immunosensing. Composition and chemical properties of the carboxyl-rich PP films deposited from a mixture of maleic anhydride and acetylene were investigated. Changes in the films stored in air, water, and buffer were studied and the involved chemical changes were described. Performance in SPR immunosensing was evaluated on interactions of human serum albumin (HSA) with a specific monoclonal antibody. The comparison with the mixed self-assembled monolayer of mercaptoundecanoic acid and mercaptohexanol (MUA/MCH) and one of the most widely used surfaces for SPR, the 2D and 3D carboxymethylated dextran (CMD), was presented to show the efficacy of plasma polymerized matrix layers for biosensing. The PP film-based SPR immunosensor provided a similar detection limit of HSA (100ng/mL) as MUA/MCH- (100ng/mL) and 3D CMD (50ng/mL)-based sensors. However, the response levels were about twice higher in case of the PP film-based immunosensor than in case of MUA/MCH-based alternative. The PP film surfaces had similar binding capacity towards antibody as the 3D CMD layers. The response of PP film-based sensor towards HSA was comparable to 3D CMD-based sensor up to 2.5μg/mL. For the higher concentrations (> 10μg/mL), the response of PP film-based immunosensor was lower due to inaccessibility of active sites of the immobilized antibody inside the flat PP film surface. We have demonstrated that due to its high stability and cost-effective straightforward preparation, the carboxyl-rich PP films represent an efficient alternative to self-assembled monolayers (SAM) and dextran-based layers in label-free immunosensing. Graphical abstract.

Improved antibody loading on self-assembled graphene oxide films for using in surface plasmon resonance immunosensors

Immunoassays are important for the diagnosis of many diseases. Efficient methods for the immobilization of the biorecognition elements is a relevant subject because the antibody loading is directly related to the detection range and sensitivity. Here different self-assembled films were explored to take advantage of polar oxygenated groups from graphene oxide (GO) structure. Surface plasmon resonance (SPR) sensors were modified with different GO self-assembled films and compared to the well-established thiol-functionalization with 11-MUA (11- mercaptoundecanoic acid). Self-assembled cysteamine/GO functionalized sensor (Cys-GO SA ) provided the highest protein loading (7.66 × 10 −12 mol cm −2 against 1.29 × 10 −11 mol cm −2 for the traditional thiol-functionalization). The Langmuir-Blodgett (LB) films of GO were composed of aggregates and empty spaces, which hindered the antibody anchoring (around 6.38 × 10 −12 to 3.45 × 10 −12 mol cm −2 ). The SPR response of the Cys-GO SA -modified sensor to the 4.0 ng mL −1 PSA was 125% higher than the thiol-functionalized sensor at the same concentration. This result indicates a simple and promising surface modification strategy for many important applications, not limited to SPR sensors, but for all immunoreaction-based assays as enzyme-linked immunosorbent assay (ELISA), electrochemical (e.g. amperometric and impedimetric) and quartz crystal microbalance (QCM) based sensors. • Different self-assembled graphene oxide (GO) films were tested in SPR sensors. • Antibody loading was superior for the cysteamine-GO-modified surface. • 125% higher sensitivity (than thiol-method) was achieved with Cys-GO modification. • Cys-GO self-assembled film is promising for immunosensing applications.

Development of a surface plasmon resonance immunosensor and ELISA for 3-nitrotyrosine in human urine

3-Nitrotyrosine (3-NT) is thought to be a relevant biomarker of nitrosative stress which is associated with many inflammatory and chronic diseases. It is necessary to develop confidential method for specific and sensitive 3-NT detection. In this paper, on the basis of anti-3-NT specific antibody, we developed a label-free indirect competitive surface plasmon resonance (SPR) immunosensor and ELISA for the detection of 3-NT. Under the optimized conditions, the SPR immunosensor can obtain a linear range of 0.17–6.07 μg/mL and a limit of detection (LOD) of 0.12 μg/mL while the ELISA can reach 0.33–9.94 μg/mL and a LOD of 0.24 μg/mL.The selectivity of 3-NT was also testified by six kinds of amino acid analogues. Besides, the developed SPR immunosensor was compared thoroughly with a conventional ELISA in spiked analysis of urine samples. Good recoveries and correlation between these two methods were observed (R2 = 0.964). Therefore, it is concluded that the automated SPR platform can be applied to quantify 3-NT in biological samples with its sensitivity, accuracy, and real-timing.

Rapid detection of Escherichia coli using fiber optic surface plasmon resonance immunosensor based on biofunctionalized Molybdenum disulfide (MoS2) nanosheets

The molybdenum disulfide (MoS2) nanosheets functionalized fiber optic surface plasmon resonance (SPR) immunosensor has been reported for the sensitive detection of Escherichia coli (E. coli). The MoS2 nanosheets were prepared by chemical exfoliation method. The synthesised nanostructures were characterized for their structural, morphological and optical properties. The E. coli monoclonal antibodies were successfully immobilized on the MoS2 functionalized sensing platform via hydrophobic interactions. An alternative method simplifying the antibodies immobilization process by functionalization of 2D nanomaterial (MoS2 nanosheets) for rapid (~15 mins) bacterial quantification is presented in this study. The immunosensor uses wavelength interrogation method and a strong linear relationship (R2 = 0.994) was observed between spectral response of immunosensor and different concentration of E. coli. The nonspecificity and cross-reactivity studies of the developed immunosensor were investigated with detection of Salmonella Typhimurium and Staphylococcus aureus. To demonstrate the practical application, spiked samples of water and orange juice were analysed with acceptable recovery results. The label-free immunosensor exhibits better performance, detection limit (94 CFU/mL), high sensitivity (2.9 nm/1000 CFU mL−1; 3135 nm/RIU) and profound specificity as compared to conventional fiber optic SPR sensor (detection limit: 391 CFU/mL, sensitivity: 0.6 nm/1000 CFU mL−1; 1646 nm/RIU). This sensing platform shows promising applications in regular water and food quality monitoring for various pathogenic microorganisms.

PH-Based Detection of Target Analytes in Diluted Serum Samples Using Surface Plasmon Resonance Immunosensor.

Detection of minute quantities of target antigens in serum samples (consisting of a mixture of proteins/biomolecules) can be achieved by enhancement of the capture efficiencies of heterogeneous immunosensors. An important process parameter which affects the capture of target analytes in such immunosensors is the pH of the solution as the target proteins present in the serum samples are charged molecules. Here, we investigated the capture of prostate-specific antigens (PSAs), first in a mixed-analyte system wherein the solution contained two other non-specific proteins along with the target analyte, using the surface plasmon resonance spectroscopy. There are no reports on the detection of antigens in a mixed system based on the optimization of the pH values of the carrier fluid, and this is the motivation of the present work. Further, we studied interference effects caused by the presence of these non-specific proteins in the mixed-analyte systems by artificially increasing the ratio of the interfering proteins to that of the target protein. Eventually PSA spiked into the rabbit serum samples was captured through the optimization of the pH of the solution. We could detect PSA in the serum samples when diluted to 100 times or more, where the amounts of other interfering proteins were ~ 66 times that of the amount of PSA. This study proposes a heterogeneous immunosensor to detect the target analytes in the diluted serum samples by tuning pH the of solution mixture, which can be utilized to detect disease biomarkers in serum samples.

Binding properties of broad-specific monoclonal antibodies against three organophosphorus pesticides by a direct surface plasmon resonance immunosensor.

In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC50 ranging from 2.93 to 19.71ngmL-1. Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (KD) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10-9M and 4.26 × 10-9M for parathion, 6.75 × 10-9M and 4.17 × 10-9M for methyl-parathion, and 2.44 × 10-8M and 1.19 × 10-8M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs. Graphical abstract ᅟ.

Cyclopropylamine plasma polymer surfaces for label-free SPR and QCM immunosensing of Salmonella

We report on the first successful application of nitrogen-containing functional surfaces prepared by plasma polymerization in surface plasmon resonance (SPR) immunosensing. The plasma polymers (PPs) were deposited from cyclopropylamine (CPA) vapors onto gold surfaces of SPR and quartz crystal microbalance (QCM) sensors. It provided an excellent platform for a stable immobilization of antibodies using glutaraldehyde (GA) activation. The performance of the SPR immunosensor was proven by a model pair of monoclonal antibody (Ab) AL-01 and human serum albumin as well as by the selective detection of bacterial pathogen Salmonella Typhimurium using the immobilized anti-Salmonella Ab. A baseline drift of the measured SPR signal, caused by the long-term reactivity of CPA plasma polymers, was well stabilized by 18 h immersion in phosphate buffered saline prior to the GA activation. The immunosensors can be successfully regenerated several times using 10 mM NaOH. The limit of detection of 105 CFU/mL and a wide linear response were achieved for Salmonella. The assay parameters are comparable to the conventional label-free methods. However, the replacement of conventional immobilization matrix with the CPA PPs offers an advantage of excellent adhesion to gold QCM and SPR surfaces achieved by fast and eco-friendly procedure compatible with the vacuum deposition of gold.