Fluorescent Immunosensor Research Articles

Polymer brush based fluorescent immunosensor for direct monitoring of interleukin-1β in rat blood.

A sandwich immunosensor was successfully developed for monitoring of interleukin-1β (IL-1β) in rat whole blood. The substrate stainless steel (SS) was first coated with a polydopamine layer and subsequently grafted with poly(ethylene glycol) methacrylate brushes, onto which a sandwich immunosensor was modified for detection of IL-1β. The device has been successfully applied for monitoring of IL-1β with a limit of detection of 4.7 pg mL-1, and a linear detection range of 12.5-200 pg mL-1. Good specificity and selectivity for monitoring of IL-1β in rat macrophage secretion were achieved. Furthermore, this device was validated by detection of IL-1β in rat whole blood samples with greater concentrations observed in obese rats compared to control, and strong positive correlation between concentrations of IL-1β and blood glucose. These results suggest this device is feasible for direct detection of target analytes in biological samples.

CdTe@SiO2 signal reporters-based fluorescent immunosensor for quantitative detection of prostate specific antigen

In this paper, an immunosensor using CdTe@SiO2 core-shell nanoparticles as labels was constructed for highly sensitive detection of prostate-specific antigen (PSA). In this approach, CdTe@SiO2 core-shell nanoparticles were synthesized using the sol-gel method. The additional Cd ions and sulfur source in SiO2 shell can greatly enhance the fluorescence intensity of CdTe nanocrystals. The reason is the formation of CdS-like cluster in SiO2 shell, which reduced the quantum size effect. The obtained CdTe@SiO2 nanoparticles also exhibited excellent biocompatibility, which was ideal for applying in biomarker detection. Furthermore, PSA capture antibodies functionalized magnetic Fe3O4 nanoparticles (Fe3O4-Ab1) were utilized in the proposed immunosensor to capture and enrich the PSA. The captured PSA was then immuno-recognized by CdTe@SiO2 labeled with PSA detection antibodies (CdTe@SiO2-Ab2) by forming the sandwich complex Fe3O4-Ab1/PSA/Ab2-CdTe@SiO2. The construction of this immunosensor was confirmed by fluorescence spectroscopy. The proposed immunosensor showed a good linear relationship between the fluorescent intensity and the target PSA concentration ranging from 0.01 to 5 ng/mL, and a detection limit as low as 0.003 ng/mL was achieved. The sensor also exhibited good specificity to PSA. This highly sensitive and specific immunosensor has great potential to be used in other biological detection.

Serological diagnosis of Toxoplasmosis disease using a fluorescent immunosensor with chitosan-ZnO-nanoparticles

This article describes a microfluidic LIF immunosensor for the quantitative determination of anti-Toxoplasma gondii IgG (anti-T. gondii) specific antibodies. The serological detection of these antibodies plays a crucial role in the clinical diagnosis of toxoplasmosis. Zinc oxide nanoparticles (ZnO-NPs) obtained by wet chemical procedure were covered with chitosan and then used to conjugate T-gondii antigens into the central microfluidic channel. Serum samples containing anti-T-gondii IgG antibodies were injected into the immunosensor where they interact immunologically with T. gondii antigens. Bound antibodies were quantified by the addition of anti-IgG antibodies labeled whit alkaline phosphatase (ALP). ALP enzymatically converts the non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to soluble fluorescent methylumbelliferone that was measured using excitation at 355 nm and emission at 440 nm. The relative fluorescent response of methylumbelliferone is proportional to the concentration of anti-T. gondii IgG antibodies. The coefficients of variation are less than 4.73% for within-day assays and less than 6.34% for between-day assays. Results acquired by LIF immunosensor agree with those obtained by enzyme-linked immunosorbent assay method, suggesting that the designed sensor represents a promising tool for the quantitative determination of anti-T. gondii IgG antibodies of clinical samples.

A magnetic beads-based portable flow cytometry immunosensor for in-situ detection of marine biotoxin.

Okadaic acid (OA), a representative diarrhetic shellfish poisoning toxin, mainly produced by toxigenic dinoflagellates, has significant hazard to public health. Traditional methods for detection of OA can not give the consideration to the need of rapid, high sensitive, quantitative and in-situ detection at the same time. Herein, a new effective detection method of OA was developed based on fluorescence immunosensor and flow cytometry (FCM). In this assay, Streptavidin-coated magnetic beads (MBs) were used as the supporter to immobilize the biotinylated OA. Modified MBs competed with the free OA in the sample solution to bind with the anti-OA monoclonal antibody (OA-MAb). The R-phycoerythrin (R-PE) dye labeled IgG was served as a secondary antibody to perform fluorescence detection. A portable flow cytometry was applied for the in-situ fluorescence quantification. The results showed that the OA concentration was inversely proportional to the R-PE fluorescence intensity. The detection method took within 50min with a limit of detection (LOD) was 0.05μg/L and range from 0.2 to 20μg/L for OA detection. Moreover, the matrix effect and the recovery rate were assessed during real sample measurement, showing a high recovery. Performance features such as high sensitivity, low LOD, speediness and simplicity of the analysis protocol, shows this biosensing-systems as a promising tool for routine use.

Immunosensor Based on Antibody-Functionalized MoS2 for Rapid Detection of Avian Coronavirus on Cotton Thread.

Infectious bronchitis virus (IBV), an avian coronavirus, significantly affects the performance of both the egg-laying and meat-type birds causing the foremost of economic loss in poultry industry. This paper aims to develop a rapid, low-cost, and sensitive biosensor for IBV detection by using molybdenum disulfide (MoS2). MoS2 is a 2-D nanosheet which has strong high fluorescence-quenching ability when applied to a dye-labeled antibody (Ab). In this paper, we developed an Ab-functionalized MoS2-based fluorescent immunosensor, which utilized the fluorescence resonance energy transfer (FRET) between the MoS2 and fluorescence dye during the Ab–antigen interaction. The assay was performed on a low-cost cotton thread-based microfluidic platform due to the good wicking property and flexibility. Upon the optimization of assay conditions, the immunosensor demonstrated remarkable sensitivity of n}{}4.6times 10^{2}n EID50 per mL and specificity with a dynamic linear response range of 102–106 EID50 per mL for IBV standard solutions. The developed immunoassay successfully detected the IBV spiked chicken serum with satisfactory results. The foregoing presents its potential application for on-farm detection.

Development of graphite carbon nitride based fluorescent immune sensor for detection of alpha fetoprotein

A novel fluorescent immunosensor for determination of alpha fetoprotein (AFP) in serum samples has been developed based on the nano graphite carbon nitride (g-C3N4) as fluorophore and immunomagnetic beads (MBs) as separation material. The bulk g-C3N4 was obtained by thermal polymerization of melamine, and then carboxylated and exfoliated to acquire the carboxylated nano g-C3N4 (c-n-g-C3N4), which has been characterized and the results showed that it had excellent fluorescent properties. The antibodies of AFP (Ab1, Ab2) were conjugated to the MBs and the c-n-g-C3N4, respectively. In assay of AFP detection, the magnetic part of the immunosensor, MBs-Ab1, would form the sandwich type complex with the signal part of the sensor, c-n-g-C3N4-Ab2. The developed immunosensor could simplify the process of separation due to the MBs. The results illustrated that proposed approach held a good linearity between the fluorescence intensity of the sensor and the AFP concentration ranging from 5–600ng/mL with the limit of detection as low as 0.43ng/mL, and its spiking recoveries ranged from 98.2% to 105.9% with RSD from 2.1% to 3.5%. The fabricated fluorescent immunosensor possesses the merits of good sensitivity, excellent selectivity, high biocompatibility and low cost, and the results provide a novel clue to develop immunosensor for determination of the biomarkers in complex matrices.

Fluorescence immunosensor for cardiac troponin T based on Förster resonance energy transfer (FRET) between carbon dot and MoS2 nano-couple.

In this study, we demonstrated a prompt and sensitive detection technique for cardiac troponin T (cTnT) in buffer and biological fluid (serum) using an NIR-active fluorescent anti-cTnT-labelled carbon dot (CD) and molybdenum disulfide (MoS2)-based nano-couple. Exfoliated MoS2 nanosheets strongly grasp the anti-cTnT-labelled CDs over their surface, and an excited-state non-radiative energy transfer mechanism takes place from CDs to MoS2, thereby quenching the upconversion fluorescence. The nonlinear and upward Stern-Volmer relationship is observed, which indicates a combined static and dynamic quenching. Static and time-resolved fluorescence measurements predict distance-dependent Förster resonance energy transfer (FRET) dynamics, which control the detection process. In the presence of cTnT, the energy transfer process gets hindered due to strong antibody/antigen (anti-cTnT/cTnT) interaction. The cTnT molecules affect the positions of the nano-couple and cause effective detachment of CDs from the MoS2 surface. This results hindrance in the energy transfer process with consequent restoration of upconversion intensity. A linear response is observed between the cTnT concentration and the restored fluorescence intensity in the concentration range of 0.1-50 ng mL-1 with a limit of detection of 0.12 ng mL-1 and a limit of quantification of 0.38 ng mL-1. Statistical analysis shows that the present assay possesses an accuracy of 101.4 ± 3.76 with a co-relation co-efficient of 0.99. Thus, CD/MoS2 provides a promising platform for the sensitive detection of cTnT.

Protein-responsive rolling circle amplification as a tandem template to drive amplified transduction of fluorescence signal probes for highly sensitive immunoassay.

Proximity binding-induced DNA assemblies possessing outstanding flexibility and diversity can provide great accessibility for output translation of amplifiable DNA probes or directly detectable signals. Based on a novel protein-responsive DNA assembly constructed via proximity binding-initiated rolling circle amplification (RCA), we present a sensitive and specific fluorescence resonance energy transfer (FRET) strategy for the immunoassay of mucin 1 (MUC1). The resultant RCA product containing numerous and continuous repeats as the tandem template accelerates the transduction of signal probes through the disassembly of triple-helix stem structure (THSS), achieving a significantly amplified FRET signal readout.

Immunofluorescence–based biosensor for the determination of dengue virus NS1 in clinical samples

The sharp increase in incidence of dengue infection has necessitated the development of methods for the rapid diagnosis of this deadly disease. Here we report the design and development of a reliable, sensitive, and specific optical immunosensor for the detection of the dengue nonstructural protein 1 (NS1) biomarker in clinical samples obtained during early stages of infection. The present optical NS1 immunosensor comprises a biosensing surface consisting of specific monoclonal NS1 antibody for immunofluorescence-based NS1 antigen determination using fluorescein isothiocyanate (FITC) conjugated to IgG antibody. The linear range of the optical immunosensor was from 15−500ngmL−1, with coefficient of determination (R2) of 0.92, high reproducibility (the relative standard deviation obtained was 2%), good stability for 21days at 4°C, and low detection limit (LOD) at 15ngmL−1. Furthermore, the optical immunosensor was capable of detecting NS1 analytes in plasma specimens from patients infected with the dengue virus, with low cross−reaction with plasma specimens containing the Japanese encephalitis virus (JEV) and Zika virus. No studies have been performed on the reproducibility and cross-reactivity regarding NS1 specificity, which is thus a limitation for optical NS1 immunosensors. In contrast, the present study addressed these limitations carefully where these two important experiments were conducted to showcase the robustness of our newly developed optical-based fluorescence immunosensor, which can be practically used for direct NS1 determination in any untreated clinical sample.

Integrated optical waveguide-based fluorescent immunosensor for fast and sensitive detection of microcystin-LR in lakes: Optimization and Analysis

Nowadays, biosensor technologies which can detect various contaminants in water quickly and cost-effectively are in great demand. Herein, we report an integrated channel waveguide-based fluorescent immunosensor with the ability to detect a maximum of 32 contaminants rapidly and simultaneously. In particular, we use waveguide tapers to improve the efficiency of excitation and collection of fluorescent signals in the presence of fluorophore photobleaching in a solid surface bioassay. Under the optimized waveguide geometry, this is the first demonstration of using such a type of waveguide immunosensor for the detection of microcystin-LR (MC-LR) in lake water. The waveguide chip was activated by (3-Mercaptopropyl) trimethoxysilane/N-(4-maleimidobutyryloxy) succinimide (MTS/GMBS) for immobilization of BSA-MC-LR conjugate, which was confirmed to have uniform monolayer distribution by atomic force microscopy. All real lake samples, even those containing MC-LR in the sub-microgram per liter range (e.g. 0.5 μg/L), could be determined by the immunosensor with recovery rates between 84% and 108%, confirming its application potential in the measurement of MC-LR in real water samples.

An amplified comparative fluorescence resonance energy transfer immunosensing of CA125 tumor marker and ovarian cancer cells using green and economic carbon dots for bio-applications in labeling, imaging and sensing

CA125, is a marker in the clinical diagnosis of several cancers and currently is the best serum-based tumor marker for ovarian cancer. Here, we developed an ultrasensitive antibody-ssDNA aptamer sandwich-type fluorescence immunosensor for CA125 detection. Based on a novel signal amplification strategy the carbon dots (CDs) functionalized with aptamer (CD-aptamer) used as detection probe and PAMAM-Dendrimers/AuNPs was used for covalent attachment of CA125-antibody and completing the sandwich assay method. By measuring of fluorescence resonance energy transfer (FRET) signals between CDs and AuNPs as nanoquenchers, the fluorescence signal quenched during sandwich complex formed between anti-CA125, CA125 and CDs-Aptamer and decreasing of fluorescence response signal is related to CA125 concentrations. Under optimal conditions, the immunosensor exhibited an extremely low calculated detection limit of 0.5fg/mL with wide linear range 1.0fg/mL to 1.0ng/mL of CA 125. The application of the immunosensor for CA125 detection in serum samples and measuring of ovarian-cancer cells was also investigated. The immunosensor revealed good sensitivity and specificity with ovarian cell concentrations from 2.5×103 to 2×104cells/mL with correlation coefficient of 0.9937 and detection limit of 400cells/mL (4 cell in 10μL), indicating potential application of immunosensor in clinical monitoring of tumor biomarkers. Furthermore, the cell viability was not changed upon treatment with CDs probe during 24h, showing the low cytotoxicity of the probe. More importantly, CDs-antibody hybrid was achieved in selective imaging of the cancer cells over the OVCAR-3 line cells, implying its potential applications in biosensing, as well as in cancer diagnosis.

Fluorescence Immunosensing of Insulin via Protein Functionalized Gold Nanoclusters.

A novel fluorescent immunosensor for the determination of insulin, an important peptide hormone, has been fabricated in homogeneous solution. Bovine serum albumin (BSA) capped gold nanoclusters (AuNCs), being highly biocompatible, were used to label the insulin antibody (Ab). In presence of the target antigen, insulin (Ag), specific immunoreaction between Ab and Ag takes place leading to the fluorescence recovery of AuNCs that provided signal readout for the immunosensing process. A linear relationship between the fluorescence signal and concentration of insulin was obtained in the range of 4.9×10-9g/mL to 3.8×10-8g/mL, with a detection limit of 1.1×10-10g/mL. Furthermore, application of the present approach in human insulin injection and synthetic blood serum has been demonstrated, thus promising an efficient platform for clinical diagnostics.

A Novel Quantum Dot Fluorescence Immunosensor Based on Magnetic Beads and Portable Flow Cytometry for Detection of Okadaic Acid

Okadaic acid (OA) is a representative diarrhetic shellfish poisoning toxin which is highly toxic and carcinogenic to human, and rich in polluted shellfish. In this work, a novel and high sensitive fluorescence immunosensor based on magnetic beads (MBs) and quantum dots (QDs) had been developed for OA detection. Carboxylic acid modified MBs were used as supporter to immobilize the OA-BSA, competed with the OA in the sample solution to bind with the anti-OA monoclonal antibody (OA-MAb). The CdTe QDs (EX600 nm) labeled IgG was served as secondary antibody to perform fluorescence detection. A portable flow cytometry, Moxi Flow was applied for OA on-site quantification. The results showed that the OA concentration was inversely proportional to the QDs fluorescence intensity. The limit of detection (LOD) was 0.05 μg/L with a linear range of 0.2–20 μg/L for OA detection, which was far lower than traditional id-ELISA strategy. Moreover, OA detection for the real sample could be completed within 1 hour. The matrix effect and the recovery rate were also assessed during real sample measurement, showing a high recovery.

A sensitive immunosensor for microcystin-(leucine–arginine) based on fluorescent self-assembled multilayers

In this paper, a novel fluorescent immunosensor based on self-assembled multilayers was constructed to detect microcystin-(leucine–arginine).

Fluorescent immunosensor using AP-SNs and QDs for quantitation of IgG anti-Toxocara canis

The objective of this work was to develop a microfluidic immunosensor for the quantitative determination of IgG antibodies to Toxocara canis (IgG anti-T. canis), causal agent of toxocariasis. This disease is caused by accidental ingestion of infective eggs that hatch into the first portion of the intestine. Subsequently, the juvenile stages are distributed throughout the body, generating symptoms from mild to severe manifestations. The possibility of early diagnosis is of great importance, allowing proper management and treatment of patients suffering from toxocariasis.IgG anti-T. canis antibodies detection was carried out using a non-competitive immunoassay, in which excretory-secretory antigens from T. canis second-stage larvae (TES) were covalently immobilized on 3-aminopropyl-functionalized silica-nanoparticles (AP-SNs). Antibodies present in serum samples immunologically reacted with TES and then were quantified by using a second antibody labeled with cadmium selenide zinc sulfide quantum dots (CdSe-ZnS QDs). The concentration of IgG anti-T. canis antibodies present in the serum sample was measured by LIF detector, using excitation lambda at 491nm and emission at 540nm. The total assay time was 30min, having made LIF detection in less than 1min. The detection limit calculated for the proposed methodology was 0.12ngmL−1 and the coefficients of intra- and inter-assay variation were less than 6%. The results show the usefulness of the developed immunosensor for the fast determination of IgG antibodies anti-T. canis.

A fluorescent immunosensor for high-sensitivity cardiac troponin I using a spatially-controlled polymeric, nano-scale tracer to prevent quenching

For detection of high-sensitivity cardiac troponin I (hs-cTnI<0.01ng/mL), signal amplification was attained using a rapid immunosensor with a fluorescently-labeled, polymeric detection antibody. As fluorescent molecules tend to quench when they are less than 10nm apart, a synthetic scheme for the labeled antibody was devised to control the molecular distance and so minimize the quenching effect in a single conjugate unit. To this end, we first performed novel polymerization of fluorophore-coupled streptavidin (FL-SA) with biotinylated detection antibody (b-Ab) in a stepwise manner by adding FL-SA to b-Ab five times sequentially. Relative spatial positions of the fluorophore molecules in the polymer were then distally fixed using di-biotinylated oligonucleotides and passed through a 0.45µm filter to obtain a polymer of uniform size (i.e., ~400nm in diameter). We produced polymeric tracers using two different inexpensive fluorophores, Dylight 650 and Alexa 647, and applied it to the detection of hs-cTnI spiked in human serum using a two-dimensional chromatography-based immunosensor. The tracers showed a limit of detection of 0.002ng/mL for Dylight 650 and 0.007ng/mL for Alexa 647. The standard curves linearized via log-logit transformation exhibited regression lines with correlation coefficients (R2)>0.97. The total coefficient of variation for the overall standard curve was 3.4±3.3% for the Dylight fluorophore and 5.9±1.5% for the Alexa dye. Such performances were comparable to those of the reference systems employing sophisticated technologies, Pathfast (Mitsubishi, Japan) and i-STAT (Abbott, US), with a strong correlation (R2>0.91) for the concentration range <100pg/mL.

Quantitative nanoimmunosensor based on dark-field illumination with enhanced sensitivity and on–off switching using scattering signals

A nanoimmunosensor based on wavelength-dependent dark-field illumination with enhanced sensitivity was used to detect a disease-related protein molecule at zeptomolar (zM) concentrations. The assay platform of 100-nm gold nanospots could be selectively acquired using the wavelength-dependence of enhanced scattering signals from antibody-conjugated plasmonic silver nanoparticles (NPs) with on–off switching using optical filters. Detection of human thyroid-stimulating hormone (hTSH) at a sensitivity of 100zM, which corresponds to 1–2 molecules per gold spot, was possible within a linear range of 100zM–100fM (R=0.9968). A significantly enhanced sensitivity (~4-fold) was achieved with enhanced dark-field illumination compared to using a total internal reflection fluorescence immunosensor. Immunoreactions were confirmed via optical axial-slicing based on the spectral characteristics of two plasmonic NPs. This method of using wavelength-dependent dark-field illumination had an enhanced sensitivity and a wide, linear dynamic range of 100zM–100fM, and was an effective tool for quantitatively detecting a single molecule on a nanobiochip for molecular diagnostics.

Kinetic analysis of a high-affinity antibody/antigen interaction performed by planar waveguide fluorescence immunosensor

Methods based on optical biosensors for the investigation of biomolecular interactions between high-affinity antibodies and antigens has advanced over the last years.

Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI)

Mycotoxins and industrial chemicals, such as aflatoxin M1 and melamine, now commonly exist in milk and cause potential health risks. This study presents an indirect competitive immunoassay through multiplex planar waveguide fluorescence immunosensor (MPWFI) for rapid, sensitive, and simultaneous detection and quantification of aflatoxin M1 and melamine by applying the principle of immunoreaction and total internal reflect fluorescent.Double-channel standard curves with appropriate logistic correlation (R2>0.99) were plotted, respectively. The working ranges (0.073–0.400ng/mL and 26.38–270.00ng/mL, respectively) were calculated, as well as the limit of detection (0.045 and 13.37ng/mL, respectively), when two analytes were simultaneously detected. Both results satisfied the requirements for the maximum amount set by the WHO, which illustrated that the current method was better than some other standard methods. The recovery rates in the actual samples ranged from 85% to 103%, with relative standard deviations between 1.3% and 6.5%, which indicated high accuracy and repeatability.

Two-dimensional paper chromatography-based fluorescent immunosensor for detecting acute myocardial infarction markers.

A novel washing scheme following antigen-antibody reactions with analyte was used during construction of a fluorescent immunosensor to resolve the background problem in the lateral flow assay with human serum. An immuno-membrane strip was devised to simultaneously measure cardiac troponin I (cTnI), creatinine kinase-MB isoform (CK-MB), and myoglobin to diagnose acute myocardial infarction. This strip was then installed within a cartridge containing a built-in washing solution tank, which was used to supply the solution across the signal generation pad of the strip after the immune reactions. Such cross-flow washing was initiated by onset-signaling from the internal control and began to run automatically upon sample addition. Under optimal conditions, the immunosensor displayed a stably suppressed background baseline, enabling us to attain a low detection limit for cTnI (0.05 ng/mL) as well as favorable reproducibility for repetitive measurements (relative standard deviation <10%). No interference was observed among the different complex formations at the respective analyte sites, and no artifacts were caused by sample matrices. We tested the performance relationship with the Pathfast reference system for positive serum samples (36 for cTnI, 58 for CK-MB, and 17 for myoglobin), and the correlation coefficients were >0.98. This result suggests that the new immunosensor system based on two-dimensional chromatography can be used for clinical testing.