Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.     

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody-glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.     

Activation of mouse macrophages by muramyl dipeptide coupled with an anti-macrophage monoclonal antibody.

A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues were prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules. Mouse peritoneal macrophages, incubated for 24 h with MDP-mAb3A33 conjugate became cytostatic against P815 mastocytoma cells, whereas unconjugated mAb3A33 and MDP-bound to a nonspecific rat IgG2a were ineffective. An enhancement of the cytostatic activity induced by MDP-mAb3A33 conjugate was obtained in the presence of gamma-IFN. These results show that several tens of MDP molecules can be linked to a macrophage-specific monoclonal antibody by using a neutral intermediate polymer without impairing the binding antibody capacity and that this type of MDP conjugate can efficiently activate macrophages and therefore could be the basis of the development of new antitumor therapy.     

Charge-dependent sidedness of cytochrome P450 forms studied by quartz crystal microbalance and atomic force microscopy

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

Characterization of QCM sensor surfaces coated with molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolol in aqueous buffer.     

Ultrasensitive electrochemical immunosensing using magnetic beads and gold nanocatalysts

In the current study, we developed a nanocatalyst-based electrochemical immunoassay using magnetic beads (MBs) and gold nanocatalysts (AuNs). The MBs conjugated with IgG allow easy separation of target proteins and rapid immunosensing reaction, and the AuNs conjugated with IgG amplifies electroactive species via catalytic reaction of AuNs. An antimouse IgG-MB conjugate and an antimouse IgG-AuN conjugate sandwich a target mouse IgG with low nonspecific binding. Thus formed immunosensing complex is strongly attracted to an indium tin oxide (ITO) electrode modified with partially ferrocenyl-tethered dendrimers (Fc-Ds) by using an external magnet. The AuN of the immunosensing complex produces p-aminophenol from p-nitrophenol by catalytic reduction in the presence of NaBH(4), and the generated p-aminophenol is electrooxidized at the Fc-D-modified ITO electrode. The oxidized product, p-quinone imine, is reduced back to p-aminophenol by NaBH(4) and then re-electrooxidized at the electrode. This redox cycling greatly amplifies the electrochemical signal. Moreover, the Fc-D-modified ITO electrode exhibits a low background current. Accordingly, the high signal-to-background ratio allows an extremely low detection limit of 1 fg/mL (7 aM) in cyclic voltammetric experiments and, importantly, 100 ag/mL (0.7 aM) in differential pulse voltammetric experiments.     

Ferrocene-avidin conjugates for bioelectrochemical applications.

Coupling of ferrocene moieties to avidin via a flexible spacer molecule yields a conjugate which combines the unique biotin-binding properties of avidin with the reversible redox characteristics of ferrocenes. Synthesis of the conjugate has been optimised and the conjugates were characterised bio- and electrochemically. Covalent immobilisation of the conjugate on gold electrodes in a dense monolayer results in electrodes with a high binding capacity for biotinylated molecules as well as good electron transfer properties. The application potential of such electrodes for bioelectrochemical systems is demonstrated by electrochemical reduction of hydrogen peroxide under mild conditions catalysed by a bound biotin-microperoxidase MP11 conjugate.     

Highly sensitive amperometric detection of genomic DNA in animal tissues

A simple and highly sensitive method for the detection of genomic DNA in tissue samples is described. It is based on amperometric detection of target DNA by forming an analyte/polymeric activator bilayer on a gold electrode. The biotinylated target DNA is hybridized to oligonucleotide capture probes immobilized on the gold electrode, forming the first layer. A subsequent binding of glucose oxidase– avidin conjugate to the target DNA and the introduction of a second layer of a redox polymer to the electrode, via layer-by-layer electrostatic self-assembly, allow for electrochemical detection of the catalytic oxidation current of glucose in a PBS solution. Less than 2.0 fg of rat genomic DNA, for both regulated and house-keeping genes, can be easily detected in 2.5 µl droplets. The proposed procedure shows very high specificity for genomic DNA in a RT–PCR mixture.     

Synthesis of bilirubin imprinted polymer thin film for the continuous detection of bilirubin in an MIP/QCM/FIA system

A bilirubin imprinted polymer (BIP) was coated on a thiol pretreated Au electrode on a quartz crystal microbalance (QCM) chip. The BIP thin film was synthesized using 4-vinylpyridine (4-Vpy) as the monomer, divinylbenzene (DVB) as the cross-linker, and benzophenone as the initiator. By using a photo-graft surface polymerization technique with irradiation by ultra-violet (UV) light, a thin BIP film was prepared, from which a biomimetic sensor for the detection of bilirubin was developed. The sensor was able to discriminate bilirubin in solution owing to the specific binding of the imprinted sites. The BIP/QCM chip has been repeatedly used for more than 7 months in many continuous experiments. The detection signal of bilirubin from the BIP thin film/QCM was compared with the non-BIP thin film/QCM. Biliverdin, an analogue of bilirubin, was used for comparison. The analogue comparison confirmed the binding specificity of the BIP film toward bilirubin. The selectivity can be as high as 31.2. The effect of pH on the detection of bilirubin is also discussed. With proper solvent for elution and recovery, flow injection analysis (FIA) could be applied to the system. The performance of the BIP/QCM chip was evaluated. A linear calibration of the bilirubin concentration with respect to the frequency shift was successfully obtained. The reproducibility of measurements from the same BIP/QCM chip was confirmed. In addition, repeatability of detection was also confirmed from different BIP/QCM chips. In conclusion, a combined BIP thin film/QCM/FIA method was successfully established for the detection of bilirubin concentration using a molecularly imprinted film.     

Detection of estradiol at an electrochemical immunosensor with a Cu UPD|DTBP-Protein G scaffold

A copper monolayer was formed on a gold electrode surface via underpotential deposition (UPD) method to construct a Cu UPD|DTBP-Protein G immunosensor for the sensitive detection of 17β-estradiol. Copper UPD monolayer can minimize the non-specific adsorption of biological molecules on the immunosensor surface and enhance the binding efficiency between immunosensor surface and thiolated Protein G. The crosslinker DTBP (Dimethyl 3,3'-dithiobispropionimidate · 2HCl) has strong ability to immobilize Protein G molecules on the electrode surface and the immobilized Protein G provides an orientation-controlled binding of antibodies. A monolayer of propanethiol was firstly self-assembled on the gold electrode surface, and a copper monolayer was deposited via UPD on the propanethiol modified electrode. Propanethiol monolayer helps to stabilize the copper monolayer by pushing the formation and stripping potentials of the copper UPD monolayer outside the potential range in which copper monolayer can be damaged easily by oxygen in air. A droplet DTBP-Protein G was then applied on the modified electrode surface followed by the immobilization of estradiol antibody. Finally, a competitive immunoassay was conducted between estradiol-BSA (bovine serum albumin) conjugate and free estradiol for the limited binding sites of estradiol antibody. Square wave voltammetry (SWV) was employed to monitor the electrochemical reduction current of ferrocenemethanol and the SWV current decreased with the increase of estradiol-BSA conjugate concentration at the immunosensor surface. Calibration of immunosensors in waste water samples spiked with 17β-estradiol yielded a linear response up to ≈ 2200 pg mL(-1), a sensitivity of 3.20 μA/pg mL(-1) and a detection limit of 12 pg mL(-1). The favorable characteristics of the immunosensors such as high selectivity, sensitivity and low detection limit can be attributed to the Cu UPD|DTBP-Protein G scaffold.     

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.     

Photo-immobilization of biological components on gold-coated chips for measurements using surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM)

The photo-immobilization technique is useful for immobilization of various biomolecules on assorted material surfaces, independent of the organic functional groups that may be present. Here, we report a convenient new photo-immobilization technique that was developed by combining a nonbiofouling polymer containing polyethylene glycol and a photoreactive crosslinker for surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. By this method, nonspecific interactions were reduced and various types of molecules, bovine serum albumin, heparin, dsDNA, phosphatidylserine, Tobacco Mosaic Virus, and norfloxacine, were immobilized on an alkane thiol-modified gold surface by a single method. The interactions of photo-immobilized biomolecules and their corresponding antibodies were investigated by SPR and QCM. In addition, SPR imaging was possible using the present method.     

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.     

Quartz crystal microbalance bioaffinity sensor for biotin based on mixed self-assembled monolayers and metastable molecular complex receptor

A quartz crystal microbalance (QCM) sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer (SAM) of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM, and enhances the amount of the immobilized molecules on the QCM, as a longer "space arm" in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017-1.67 microg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.     

An FET-type charge sensor for highly sensitive detection of DNA sequence

We have fabricated an field effect transistor (FET)-type DNA charge sensor based on 0.5 microm standard complementary metal oxide semiconductor (CMOS) technology which can detect the deoxyribonucleic acid (DNA) probe's immobilization and information on hybridization by sensing the variation of drain current due to DNA charge and investigated its electrical characteristics. FET-type charge sensor for detecting DNA sequence is a semiconductor sensor measuring the change of electric charge caused by DNA probe's immobilization on the gate metal, based on the field effect mechanism of MOSFET. It was fabricated in p-channel (P) MOSFET-type because the phosphate groups present in DNA have a negative charge and this charge determines the effective gate potential of PMOSFET. Gold (Au) which has a chemical affinity with thiol was used as the gate metal in order to immobilize DNA. The gate potential is determined by the electric charge which DNA possesses. Variation of the drain current versus time was measured. The drain current increased when thiol DNA and target DNA were injected into the solution, because of the field effect due to the electrical charge of DNA molecules. The experimental validity was verified by the results of mass changes detected using quartz crystal microbalance (QCM) under the same measurement condition. Therefore it is confirmed that DNA sequence can be detected by measuring the variation of the drain current due to the variation of DNA charge and the proposed FET-type DNA charge sensor might be useful in the development for DNA chips.     

Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

Endotoxin detection in a competitive electrochemical assay: synthesis of a suitable endotoxin conjugate

A biotin-lipopolysaccharide (biotin-LPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng(-1). The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotin-LPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidin-horseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotin-LPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml(-1), equivalent to 0.07 EU ml(-1).     

Quartz crystal microbalance immunosensors for environmental monitoring

This paper presents discussion of quartz crystal microbalance (QCM) immunosensors for environmental monitoring. Factors limiting the practical application of antibodies to analytical problems are also presented. Among several candidates for the QCM immunosensor device, selected QCM devices and oscillating circuits were tested thoroughly and developed to obtain highly stable and sensitive frequency signals. The biointerface of QCM immunosensor was designed and controlled to immobilize antibody on the QCM surface, to reduce non-specific binding and to suppress denaturation of immobilizing antibody by self-assembled monolayer technique and artificial phospholipid (2-methacryloyloxyethyl phosphorylcholine (MPC)) polymer. MPC polymer as a antibody-stabilizing reagent was added to reduce non-specific binding of the antigen solution and stabilize the immunologic activity of the antibody-immobilized QCM. In addition, it provides examples for detection and quantitation of environmental samples using QCM immunosensors. The analytical results for fly ash extracted samples of dioxins using the QCM immunosensor indicated a good relationship with GC/MS methods. The integrating protocols of the competitive immunoassay and signal-enhancing step are for detecting low molecular analytes with extremely low detection limits using an QCM immunosensor. Furthermore, its detect limitation was extended from 0.1 to 0.01 ng/ml by the signal-enhancing step when the anti-bisphenol-A antibody conjugated MPC polymeric nanoparticles was used. The QCM immunosensor method has demonstrated its effectiveness as an alternative screening method for environmental monitoring because these results were compared with results obtained through environmental monitoring methods such as ELISA and GC/MS.