Sensors competitive assay

Fig. I Dependence of response of anisotropy sensor on analyte concentration in direct and competition assays (a) and this dependence for direct assay at different correlations between cp and xF (b)...

Fig. 37 (a) QD-based sensing of cocaine by the formation of a cocaine-aptamer supramolecular structure that triggers FRET and (b) time-dependent luminescence spectra of the system in the presence of cocaine. The inset shows a calibration curve for variable concentrations of cocaine and a fixed so observation time of 15 min. (c) Schematic of the FRET-based TNT sensor and (d) increase of the QD luminescence upon addition of TNT in the competitive assay format. (Reprinted with permission from [220, 221], Copyright 2009 Royal Society of Chemistry and 2005 American Chemical Society)... 

S. O. Obare and C. J. Murphy, A Two-Color Fluorescent Lithium Ion Sensor, Inorg. Chem. 2001,40, 6080 L. Fabbrizzi, N. Marcotte, F. Stomeo, and A. Taglietti, Pyrophosphate Detection in Water by Fluorescence Competition Assays, Angew. Chem. Int. Ed 2002,41, 3811. 

FIGURE 2 Detection mechanisms in SPR-based sensors. (A) Direct assay direct detection of analyte. (B) Competitive assay analyte competes with an internal standard. (C) Sandwich assay analyte is trapped between two antibodies. (D) Inhibition assay analyte is pretreated with an antibody. 

Fig. 5. Example of competitive and displacement assays. (A) Competitive assay of atrazine, binding curves obtained for mixtures of MAb (ascites fluid, 1,000x diluted) preincubated for 15 min with variable concentrations of atrazine. (B) Displacement of antibody from the sensor in the presence of increasing concentrations of the analyte -peptide representing a surface epitope of the antigen. The antigen-modified sensor was preincubated with antibody (not shown), thus a constant amount of immunocomplexes (fa 350 Hz) was present before starting each of the measurements.

The direct interaction of small organic molecules with receptors immobilized on the sensor surface is difficult to detect. Two approaches may be used to detect or quantify the presence of small analytes in a process stream, displacement assays or competition assays. 

Detection of analytes can be performed using either direct detection methods or indirect detection methods. In the case of direct detection methods, an analyte or parts of an analyte are bound to the sensing surface producing the sensor response. Direct detection methods include direct detection of the analyte, sandwich assays, and competitive assays. In indirect detection methods, the analyte induces a change in the state of a secondary system component, which subsequently induces a sensor response. The most commonly used indirect detection method is the inhibition assay. 

Competitive assays, as seen in Fig. 5b, are based on two analytes competing for the same recognition site at the sensor surface. One of the analytes is free and the other is typically conjugated to a larger protein, usually bovine serum albumin or casein. The concentration of the conjugated analyte is fixed from solution to solution. The two analytes are mixed in a solution and passed across the sensing surface. The sensor response will be inversely proportional to the concentration of analyte in the target solution. 

In an inhibition assay, as seen in Fig. 5c, the analyzed sample is preincubated with an antibody for the targeted analyte. Subsequently, the mixture is injected in the SPR sensor with an analyte derivative immobihzed on the sensor surface and the binding of the unreacted antibody to the analyte derivative is measured. As with the competitive assay, the sensor response is inversely proportional to the concentration of target analyte in the incubation solution. 

Detection of medium-sized and large analytes (> 10 000 Da) is usually performed directly [37,58]. As direct binding of low molecular weight analytes at the sensor surface does not usually produce sufficient refractive index change, they are typically detected using a competition assay [39], sandwich assay [40], or inhibition assay [38]. 

Fig. 5 Cartoon representations of three different assays typically used in detecting analytes with an SPR biosensor, a Sandwich assay involves the capturing of analyte by a sensing element immobilized on the sensor surface. This is followed by the binding of a secondary antibody for amplification, b In a competitive assay, native analyte and analyte conjugated to a larger protein compete to bind to an immobilized sensing element on the surface, c In an inhibition assay, analyte is incubated with a fixed concentration of antibody. This incubation solution is then passed across a surface of immobilized analyte. Free antibody binds to the sensor siuface, creating an inverse relationship between concentration of analyte in the sample and sensor response...

Shimomura et al. used a Biacore 2000 SPR sensor instrument (Biacore AB, Sweden) for detection of PCB 3,3 4,4, 5-pentachlorobiphenyl [32]. They employed competition assay format and the sensor chip with polyclonal antibodies immobilized in the dextran matrix. The sample was mixed with a conjugate of PCB-horseradish peroxidase (HRP) and injected into the sensor. The presence of the analyte was detected as a decrease in binding of PCB-HRP conjugate. The detection was performed in 15 min with a detection limit of 2.5 ngmL in buffer. The sensor was demonstrated to be regenerable by 0.1 M hydrochloric acid. 

Fig. 7 Calibration curve of SPR sensor with photografted molecular imprinted polymer and competition assay [52]...

Small molecules pose a far different set of challenges for an SPR sensor than bacteria. While the diffusion rate of the small analytes is quite large, their low molecular weight does not cause a significant increase in the local refractive index near the sensing surface. Eecause of this problem, various strategies have been developed, the most common of which is the use of an inhibition or competitive assay. 

Based on the stereoselectivity of immunoglobulins, a new chiral sensor for the detection of low-molecular-weight analytes was developed. Using surface plasmon resonance detection, enantiomers of free, underivatized a-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in non-racemic mixtiues exceeds cunently available methods therefore, such immuuoseusors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry. 

Table 2 The MIP-Based Fluorescent Sensors for the Detection of Nonfluorescent Analytes Using a Fluorescently Labeled Analog in Competitive Assays...

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