Piezoelectric Immunosensor Research Articles

Direct immobilisation of antibodies on a bioinspired architecture as a sensing platform

A sensitive and selective immunosensor for the nonlabeled detection of sulfate-reducing bacteria (SRB) is constructed using a self-polymerised polydopamine film as the immobilisation platform. Self-polymerisation of dopamine is used as a powerful approach for applying multifunctional coatings onto the surface of a gold electrode. The polydopamine film is used not only as the immobilisation platform, but also as a cross-linker reagent for the immobilisation of the anti-SRB antibody. The polydopamine film is loaded with a high density of anti-SRB antibodies linked to the substrate to obtain high response signals. The formation and fabrication of the biosensor and the quantification of antibody anchoring are monitored, and SRB detection is performed by either quartz crystal microbalance (QCM) or electrochemical impedance spectroscopy (EIS). After modeling the impedance Nyquist plots of the SRB/anti-SRB/polydopamine/gold electrode for increasing concentrations of SRB, the electron transfer resistance ( R ct) is used as a measure of immunocomplex binding. The R ct is correlated with the concentration of bacterial cells in the range of 1.8 × 10 2 to 1.8 × 10 6 CFU mL −1; the detection limit is 50 CFU mL −1. This work demonstrates a new immobilisation platform for the development of a sensitive and label-less impedimetric and piezoelectric immunosensor. This immunosensor may be broadly applied in clinical diagnoses and the monitoring of water environmental pollution. The method proposed is distinct in its ease of application, use of a simple protocol, and mild reaction conditions. These allow it to be applied to a wide variety of materials.

A Simple and Sensitive Piezoelectric Immunosensor for Cholera Toxin Based on GM1‐Incorporated Liposome Agglutination

AbstractA sensitive piezoelelctric immunosensor has been developed for the direct detection of cholera toxin. The proposed technique is based on specific interface agglutination of GM1 incorporated liposome vesicles in the presence of the cholera toxin, which causes a frequency change and is monitored by a piezoelectric device. The interface agglutination reaction would take place as soon as the cholera toxin (CT) was captured by the antibodies modified on the probe surface and the GM1 incorporated liposome in the probe cell. Meanwhile, the agglutination reaction induced both the mass effect and viscoelastic effect acting on the probe surface. The results indicate that the probe signal can be observably multiplied. In addition, an optimization of liposome composition and assay medium were investigated. Moreover, no cross‐reactivates were observed with the common protein, such as goat anti‐mouse IgG (1 mg/mL), mouse IgG (1 mg/mL), goat anti‐human IgG (1 mg/mL), and thrombin (1 mg/mL). The frequency responses of the developed piezoelelctric immunosensor are linearly correlated to cholera toxin concentration in the range of 0.5 to 7.5 µg/mL with a detection limit of 0.27 µg/mL while showed no significant loss in activity over 3 d under storage at 4°C.

Surface IR immunosensors for label-free detection of benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are a family of contaminants originating from the incomplete combustion of organic compounds, present in the urban air, water, soil and also foodstuff. Due to their recognised carcinogenicity, their concentration in urban air is routinely monitored and governmental instances have set rules as regards their maximum allowable concentration. Benzo[a]pyrene (BaP) is one of the PAHs, highly toxic, whose concentration correlates well with the total PAHs contents in environmental samples; its detection by immunosensors has been rarely described because of its low molecular weight and poor solubility in water. In this paper, we report the design and testing of optical and piezoelectric immunosensors for the determination of BaP by using Polarisation-Modulation Infrared Reflection-Absorption Spectroscopy (PM-IRRAS) or Quartz Crystal Microbalance with dissipation measurements (QCM-D) as transduction techniques, and a comparison of their analytical performances. We found relevant to set up immunosensors based on a direct or indirect competitive format. BaP was detected by PM-IRRAS with both immunosensor formats with similar sensitivity, ca. 5 μM. Flow-injection piezoelectric transduction in the indirect competitive format set up led to a slightly better sensitivity than that reached by PM-IRRAS. No general conclusion about the most efficient set up could be drawn from comparing the sensitivities, but the reported data illustrate the potential of PM-IRRAS as well as flow-injection QCM-D as efficient label-free transduction techniques.

A quartz crystal microbalance-based Immunosensor for Shrimp Allergen Determination in Food

A rapid method for determination of food allergen was carried out by quartz crystal microbalance-based immunosensor in this paper, involving the use of polyclonal antibody specific to shrimp allergen based on antibody antigen reaction. Conditions of piezoelectric immunosensor for the analysis of shrimp allergen to be completed in less than 10 min were optimized. The rapid electrode modification was developed on the basis of self-assembly with 1,6-hexanedithiol (HDT) and nanogold. The optimum conditions for detection were as follows: 15 mM HDT was coated on the electrode for 90 min, and nanogold was adsorbed for 110 min at room temperature; the antibody was coated for 3 h under 4 °C, and the coating concentration for anti-shrimp antibody was 0.16 mg/mL, the detecting time was only 500s, and the standard curve between 1 and 300 μg/mL was obtained, in consistent with Sauerbrey equation. The detection limit is 0.333 μg/mL. The recovery of immunosensor was in the range of 96.53–106.22% with coefficient of variation (CV) 6.46–8.89%. Regeneration of the electrode was also studied: piranha and strong alkali and acid had obvious effect, and the electrode can be reused for more than 13 times; glycine had less regeneration capacity for 5 times, but more conveniently. The present immunosensor has showed good performance for application in detection of shrimp allergen.

Piezoelectric immunosensor based on gold nanoparticles capped with mixed self-assembled monolayer for detection of carcinoembryonic antigen

It is very important for a piezoelectric immunosensor to increase specific binding and decrease nonspecific adsorption. This study presents the development of such a piezoelectric immunosensor for the detection of carcinoembryonic antigen . An AT-cut quartz crystal's Au electrode surface was first modified with homogenous self-assembled monolayer of cysteamine (CE). Gold nanoparticles capped with mixed self-assembled monolayer of CE and MH (6-mercapto-1-haxanol) were then attached to the CE monolayer via glutaraldehyde (GA). Antibodies were immobilized onto a mixed self-assembled monolayer of CE and MH with GA as a reactive intermediate too. The binding of target antigens onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the antigen concentration. The stepwise assembly of the immunosensor was characterized by means of cyclic voltammetry technique. This immunoassay was shown to be specific and sensitive, thus providing a viable alternative to carcinoembryonic antigen detection method.

Monoclonal and polyclonal antibodies production - preparation of potent biorecognition element

Antibodies are macromolecular structures with a high affinity to a molecule called the antigen. Though the natural aim of antibodies is the protection of the body from pathogens, they have been found to play a useful part in a number of specific treatments and diagnoses. This wider role for antibodies extends to assays where antibodies provide a recognition capability improving available physical methods. Photometrical methods such as ELISA or flow cytometry and electrochemical, optical or piezoelectric immunosensors (biosensors) are typical examples. The present review summarizes facts about the parameters and the production of antibodies. The structure of immunoglobulins, and the production and purification of monoclonal and polyclonal antibodies are described in four chapters. The review was written in order to collect the available knowledge on antibodies into one study improving orientation in this field for anyone wanting to construct immunoassays.

Immunochemical methods for the detection of sulfanylamide drugs

Recent achievements in the detection of sulfanylamide drugs in environmental objects and foodstuffs are considered. These are the specific detection of individual substances; class-specific immunoassay of the whole group of sulfamide drugs; widely used ELISA methods; biosensors; fluorescence polarization immunoassay; and new promising methods for the detection of sulfamide drugs, immunochromatographic test strips, the method using molecularly imprinted polymers, piezoelectric quartz immunosensors, etc.

Use of polyclonal antibodies to ochratoxin A with a quartz–crystal microbalance for developing real-time mycotoxin piezoelectric immunosensors

A piezoelectric immunosensor was tested for ochratoxin A (OTA) mycotoxin detection through the immobilization of OTA-bovine serum albumin (OTA-BSA) conjugate on gold-coated quartz crystals (AT-cut/5 MHz). Immunoassays were performed in a flow-injection system through frequency decreases in a quartz-crystal microbalance (QCM) because of a mass increasing during immunoreaction with anti-OTA antibodies. Three immobilization procedures for OTA-BSA (direct adsorption and covalent attachment to two alkane thiol self-assembled monolayers) were characterized with QCM in real time. Covalent attachment of the OTA-BSA conjugates through gold nanoparticles was also tested for amplifying the signal. Binding of the excess of antibodies to the immobilized OTA in an indirect competitive analysis decreased linearly the resonant frequency in the range of the OTA concentration from 10 to 128 ng/mL, with a detection limit of 8 ng/mL (signal/noise ratio of 3). A pepsin 2 mg/mL (pH = 2.1) solution was used to release antigen-antibody complexes, regenerating the biorecognition surface.

Piezoelectric immunosensor with gold nanoparticles enhanced competitive immunoreaction technique for quantification of aflatoxin B1

A simple, rapid and highly sensitive piezoelectric immunosensor has been proposed and applied to detect aflatoxin B1 (AFB1). It is unlikely that direct binding of small molecules such like AFB1 to the piezoelectric sensor surface could result in a satisfactory detection limit and sensitivity. Thus, indirect competitive immunoassay technique had been used for the detection of the target and gold nanoparticles (GNP) been employed as a ‘weight label’ to the secondary antibody for amplifying the response. This method is proven in its ability to detect AFB1 down to a level of 0.01ngmL−1 in artificially contaminated milk, which is comparable to or even exceeding the sensitivity of microtitre plate ELISA. Furthermore, the frequency responses of the immunoassay are linearly correlated to the logarithm of AFB1 concentration in the range of 0.1–100ngmL−1. The sensor could be regenerated under very mild conditions simply by immersing the sensor into glycine buffer solution to desorb the combined antibody. It is found that the as-renewed sensor could be reused at least 9 runs without obvious loss of sensing sensitivity.

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 μg l −1 for carbaryl and TCP, respectively. The sensitivity attained ( I 50 value) was around 30 μg l −1 for both compounds. Linear working ranges were 15–53 μg l −1 for carbaryl and 13–83 μg l −1 for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.

A Piezoelectric Immunosensor Based on Agglutination Reaction with Amplification of Silica Nanoparticles

AbstractA simple piezoelectric immunoagglutination assay technique with antibody‐modified nanoparticles has been developed for direct quantitative detection of protein. The proposed technique is based on the specific agglutination of goat anti‐hIgG‐coated silica (or gold) nanoparticles in the presence of human immunoglobulin G (hIgG), which causes a frequency change and is monitored by a piezoelectric device. The antibody modified on the probe surface would combine with antibody‐coated nanoparticles in the presence of antigen (hIgG) when the surface agglutination reaction took place, which couples both the mass effect and viscoelastic effect acting on the probe. The results indicate that the background interference can be substantially minimized and the probe signal can be observably multiplied. In addition, the surface of the modified probe and that after combining the complex of immunoagglutination were imaged by scanning electronic microscopy (SEM). Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination enhancer and sodium chloride to control the ion‐strength was investigated. The frequency responses of the immunoagglutination assay were found to correlate well with the hIgG concentration with a detection limit of 84 ng·mL−1.

Mass-sensitive immunosensors for determining chloroacetanilide herbicides

Highly sensitive piezoelectric quartz immunosensors are proposed for the rapid selective determination of chloroacetanilide herbicides. Receptor coatings are hapten-protein conjugates immobilized on the presilanized surface of sensor silver or gold electrodes. Complementary pairs of immunoreagents ensuring the maximum analytical signal are selected on the basis of affinity constants found by the Sketchard method. The detection limits for acetochlor and alachlor are 0.02 ng/mL and that for butachlor is 0.002 ng/mL. Sensors were used to determine residual amounts of herbicides in foodstuffs and environmental objects.

Immunosensor for Detection of Inhibitory Neurotransmitter γ-Aminobutyric Acid Using Quartz Crystal Microbalance

A piezoelectric immunosensor for sensing the low molecular weight neurotransmitter gamma-aminobutyric acid (GABA), one of two major inhibitory neurotransmitters in the central nervous system, is described. The sensing interface consists of a dextran layer covalently attached to a self-assembled monolayer of thiolamine compound on the surface of gold electrodes of the crystals. The dextran layer is further modified with GABA molecules to act as the biosensing layer. The affinity binding of monoclonal anti-GABA antibody on the modified piezoelectric crystals is studied in real time without any additional labels. The equilibrium association constant, K(eq) for binding between anti-GABA antibody and GABA molecules is 14.5 microg x mL (-1). The detection limit for anti-GABA is approximately 10 nM. The sensitivity of the sensor at a concentration corresponding to half-maximal response is 13.6 ng/mL x Hz. The functionalized sensor substrate is subsequently used for competitive determination of different concentrations of free GABA (range of 5 microM-50 mM) in PBS-BSA buffer. The detection limit of the immunosensor for sensing GABA with maximum sensitivity is approximately 42 microM.

A novel piezoelectric quartz crystal immnuosensor based on hyperbranched polymer films for the detection of α-Fetoprotein

This paper reports a novel piezoelectric quartz crystal immnuosensor based on hyperbranched polymer films for the detection of α-Fetoprotein. In this strategy, the sensing interfaces consist of a primary cystamine monolayer assembled onto Au electrodes associated with the piezoelectric quartz crystal. The monolayer is further modified with a new hyperbranched polymer which was synthesized through direct polycondensation of monomer 5-[3-(4-aminophenyl) propionylamino] isophthalic acid. The detection performances of resulting immunosensor were investigated by use of the antibody-antigen model system of α-Fetoprotein (AFP), an important indicator in the diagnosis of clinical cancers. The analytical technique is characterised through the investigation of different methods of assembling the monolayers used as supports, as well as by comparing two different types of supports. It was found that the developed sensing interface could perform more effectively in antibody-antigen binding and consequently increased the sensitivity of the whole piezoelectric immunosensor. Moreover, the method should also be useful for the construction of other kind of immunosensors.

Development of a spiral piezoelectric immunosensor based on thiol self-assembled monolayers for the detection of insulin

A spiral piezoelectric (Pz) immunosensor based on thiol self-assembled monolayers (SAMs) and fabricated with a screw clamp apparatus was developed for quantitative detection of insulin in serum. Anti-human insulin antibody was first modified with sulfosuccinimidyl 6-[3'-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP) to graft the active sulfhydryl groups into antibody molecules. Then, modified antibody molecules were assembled on a gold electrode via covalent bonding to form SAMs as the recognition element of the insulin immunosensor. The response characteristics of the Pz immunosensor, including buffer solution, time-costs, reproducibility and specificity, were also investigated in detail. Coating a Au electrode with SAMs gave better results within 40 min, with a detection range of 2.5 to 80 mIU/L and a coefficient of variance (CV) less than 5%. The stability and reusability of the immunosensor was improved with a mild eluting reagent which successfully removed the bound insulin molecules from the antibody-coated crystal without affecting the immobilized antibody. A comparison study revealed that analytical results of insulin in samples obtained with this immunosensor were in good agreement with those given by the radioimmunoassay. Thus, the proposed Pz immunosensor provided a rapid, sensitive, specific, reusable and reliable alternative for the detection of insulin in clinical laboratory.

Determination of Schistosoma japonicum circulating antigens in dilution serum by piezoelectric immunosensor and S/N enhancement

A piezoelectric immunosensor assay was developed with immobilizing immunoglobulin G (IgG) as a probe to detect Schistosoma japonicum circulating antigens (SjCAg). Analytical strategy utilizes the polyclonal antibodies with broad-spectrum recognition to a complex target with high specificity. The immobilized antibodies were purified from immunized rabbit's sera ( im-S) and infected rabbit's sera ( inf-S) by S. japonicum. The detection capacities of antibodies were compared between the sera of different phenotypes and purified fractions. The sample dilution ratios were also evaluated and optimized. Additionally, the sera with a variety of infection degrees for validation could be discriminated quantitatively. The linear dose–response relationship indicates that the systematic sensitivity of this method is below 150 Hz and the lowest limit of detectable range is above 500 cercariae of S. japonicum infection for 2 weeks. The novel immunosensor technique is well potential to determine the SjCAg in serum samples for clinical diagnosis of parasitosis in early stage.

A reusable piezoelectric immunosensor using antibody-adsorbed magnetic nanocomposite

This paper reports a simple, sensitive, and reusable piezoelectric immunosensor using magnetic hydroxyapatite (HAP)/ γ-Fe 2O 3/Au nanocomposite. Use of porous HAP nanocrystals embedded with γ-Fe 2O 3 and colloidal gold nanoparticles resulted in a multifunctional HAP/ γ-Fe 2O 3/Au nanocomposite. Under optimized conditions, the biocompatible nanocomposites were exploited for direct adsorption of large quantities of rabbit anti-human immunoglobulin G antibodies (anti-hIgG) with well-preserved immunoactivity. In a homogeneous bulk solution, the hIgG analytes were captured by the anti-hIgG-derivatized immunocomposites followed by magnetic separation/enrichment onto a bovine serum albumin (BSA)-sealed QCM probe before measuring. This QCM immunosensor can quantitatively determine concentrations of hIgG ranging from ~ 20 to 800 ng/ml, with a detection limit of ~ 15 ng/ml. Moreover, regeneration of the immunosensor can be simply realized by canceling the controllable magnetic field. With the possibility of performing the analysis automatically and considering its ease of use, high sensitivity, and good reusability, this magnetic separation-assisted QCM immunosensor may have great potential to be further tailored as a general and promising alternative for a broad range of practical applications.

Determination of antibodies to Yersinia enterocolitica bacteria using a piezoelectric quartz crystal immunosensor

A gravimetric technique is proposed for the determination of antibodies to Yersinia enterocolitica serovar O: 3 using a piezoelectric quartz crystal immunosensor based on immobilized lipopolysaccharides. Conditions are optimized and a procedure is developed for the flow-injection analysis of blood serum samples using the immunosensor as the detector. The detection limit for antibodies is found to be 1.3 μg/mL, RSD is 8%, and the analytical range is 3–100 μg/mL. The rapidity (the duration of analysis was no longer than 10 min) and the direct detection of the formed agglutinate without additional labeling are the advantages of the proposed technique.

QCM‐Based Immunosensor for the Determination of Ochratoxin A

A piezoelectric immunosensor based on a competitive format was developed for determination of ochratoxin A (OTA) concentration. Surface modifications via two self‐assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 16‐mercaptohexadecanoic acid (16‐MHDA). The quartz crystal microbalance (QCM)‐based immunosensor was fabricated by immobilizing anti‐OTA antibodies onto the surface of the 16‐MHDA‐modified electrode, and allowing competition between free OTA and that conjugated with BSA to occur. The assay exhibited a working range of 50–1000 ng/mL and a detection limit of 16.1 ng/mL. Studies of interference and matrix effects were performed to evaluate the feasibility of the developed immunosensor for the direct analysis of OTA in real samples. Recoveries were conducted at 50, 200, and 1000 ng/g and were determined to be in the range of 142%–76%. The OTA assay is specific. No cross‐reactivates were observed with citrinin.

Piezoelectric immunosensor for the direct and rapid detection ofFrancisella tularensis

A novel immunosensing device based on a piezoelectric sensor for direct detection of the biological warfare agent Francisella tularensis was developed. This sensor includes mouse polyclonal antibody immobilized in a layer of protein A covalently linked to the gold electrode of the sensor. The immunosensor is able to detect F. tularensis with the limit of detection 10(5) CFU/mL with a typical measuring cycle > 5 min. The sensor was successfully evaluated for rapid detection of F. tularensis spikes in drinking water and milk; no deterioration of sensitivity in comparison with buffer solutions was observed. The proposed concept of a rapid measurement of microbial agents seems to be promising for evaluation of samples after short pre-cultivation enrichment.