Long-wavelength fluorescence polarization immunoassay: determination of amikacin on solid surface and gliadins in solution

The versatility of the fluorescence polarization immunoassay (FPIA) is increased by using two long-wavelength labels, Nile Blue and a ruthenium(II) chelate. The first label has been used to study the potential of FPIA on a solid surface using dry reagent technology. The aminoglycoside antibiotic amikacin has been used as an analyte model, and the method has been applied to the analysis of serum samples. The second label has been used to show the practical application of FPIA to the determination of macromolecules, using gliadins as an analyte model, which have been determined in gluten-free food. Very low amounts of anti-amikacin antibodies and amikacin-Nile Blue tracer were immobilized onto nitrocellulose membranes, for the development of the amikacin method, and the consumption of reagents is lower than in conventional FPIA. Only the addition of the standard or sample extract at an adequate pH is required at the analysis time. The analyte displaces the tracer from the tracer-antibody immunocomplex, obtaining a decrease in the fluorescence polarization proportional to the analyte concentration. The gliadin-Ru(II) chelate tracer shows a relatively long lifetime, which allows the observation of differences in fluorescence polarization values between the tracer-antibody complex and the tracer alone. The dynamic range of the calibration graphs for both analytes is 0.5-10 microg mL-1 and the detection limits are 0.1 and 0.09 microg mL-1 for amikacin and gliadins, respectively. The study of the precision gave values of relative standard deviations lower than 5 and 1.5% for the amikacin and gliadin methods, respectively. Amikacin was determined in human serum samples using a previous deproteinization step with acetonitrile, obtaining recovery values in the range 83.4-122.8%. The gliadin method was applied to the analysis of gluten-free food samples by using a previous extraction step. The recovery study gave values between 94.3 and 105.0%.     

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

Magnetic bead based immunoassay for autonomous detection of toxins

We are developing an automated system for the simultaneous, rapid detection of a group of select agents and toxins in the environment. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads and a photoactive porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactive group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags. Released eTags are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated in a flow-through format with higher LODs of 32 ng/mL (or 640 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.     

Detection of antigliadin autoantibodies in celiac patient samples using a cyclodextrin-based supramolecular biosensor

Celiac disease is a condition associated with the ingestion of gluten by genetically susceptible individuals. Measurement of serum antigliadin antibodies is a diagnostic tool also used as a means of monitoring a patient's compliance to a gluten-free diet. In this work, we demonstrate the applicability of an electrochemical supramolecular platform based on cyclodextrin-modified gold surfaces to detect antigliadin antibodies in real serum samples. Several support layer-biorecognition element combinations were tested in order to maximize the electrochemical response, and the assay was optimized in terms of incubation times and resistance to nonspecific interactions. The developed supramolecular biosensor was then applied to the amperometric detection of antigliadin IgA and IgG autoantibodies in real samples of celiac disease patients under follow-up treatment; the results were compared with a commercial enzyme linked immunosorbent assay (ELISA) test, and an excellent correlation was observed between both methods.     

Molecular aptamer for real-time oncoprotein platelet-derived growth factor monitoring by fluorescence anisotropy.

Monitoring proteins in real time and in homogeneous solution has always been a difficult task. We have applied a fluorophore-labeled molecular probe based on a high-affinity platelet-derived growth factor (PDGF) aptamer for the ultrasensitive detection of PDGF in homogeneous solutions. The aptamer is labeled with fluorescein to specifically bind with the PDGF protein. Fluorescence anisotropy is used for the real-time monitoring of the binding between the aptamer and the protein. When the labeled aptamer is bound with its target protein, the rotational motion of the fluorophore attached to the complex becomes much slower because of an increased molecular weight after binding, resulting in a significant fluorescence anisotropy change. Using the anisotropy change, we are able to detect the binding events between the aptamer and the protein in real time and in homogeneous solutions (detection without separation). This assay is highly selective and ultrasensitive. It can detect PDGF in the subnanomolar range. The new method for protein detection is simple and inherits all of the advantages of molecular aptamers. Efficient oncoprotein detection using aptamer-based fluorescence anisotropy measurement will find wide applications in protein monitoring, in cancer diagnosis as well as other studies in which protein analysis is important.     

Detection methodology based on target molecule-induced sequence-specific binding to a single-stranded oligonucleotide

We have recently developed a mix-and-read format homogeneous antigen peptide based assay for detection of the antibodies (Tian, L.; Heyduk, T. Anal. Chem. 2009, 81, 5218-5225) that employed for target detection a simple biophysical mechanism of target antibody induced annealing between two complementary oligonucleotides attached to the antigen peptide. In this work, we propose and experimentally validate an alternative variant of this assay format in which target antibody binding to antigen peptide-oligonucleotide conjugate produces a complex with high sequence-specific binding affinity to a single-stranded capture oligonucleotide. This new assay format can be used for preparing various solid-surface based assays by immobilizing the capture oligonucleotide. This assay design is not limited to antibody detection. We demonstrate that it can also be employed for detecting proteins or pathogenic bacteria using oligonucleotide-labeled antibodies as target recognition elements. Preparation of these solid-surface based assays is simplified because all interactions with the solid surfaces are mediated by well-understood oligonucleotide-oligonucleotide interactions and because of the relative ease of immobilizing oligonucleotides on various solid surfaces. These unique aspects of the assay design also allow microarray-style multiplexing that could be most useful for multiplexed antibody profiling for diagnosis and analysis of cancer, autoimmune, and infectious diseases.     

An attempt to develop surface plasmon resonance based immunosensor for Karnal bunt (Tilletia indica) diagnosis based on the experience of nano-gold based lateral flow immuno-dipstick test

Karnal bunt (KB) of wheat incited by Tilletia indica is an economically important quarantined fungal pathogen. Similarity in teliospore configuration makes it difficult to differentiate KB teliospores from the teliospores of other bunt fungi using conventional approaches. In order to determine the correct identity of KB teliospore— an infectious entity, it is essential to develop specific diagnostic probes and high quality of immunological reagents against infectious entities. The immuno-dipstick assay developed in our lab is quite sensitive to detect the antigens of even five teliospores. For on-site testing of KB, a nano-gold based lateral flow immuno-dipstick assay (LFID) was also developed in our lab using anti-teliospore antibodies. For development of LFID, colloidal gold was prepared and conjugated with anti-teliospore antibodies which were further characterized. However, species specific detection is yet to be achieved by generation of monoclonal antibodies against diagnostic antigens (34 and 66 kD teliospore's proteins). Based on the experience of nano-gold based LFID, we are proposing to develop surface plasmon resonance (SPR) based label-free detection system. The same may be employed not only for determination of the quality of immunological reagents in terms of sensitivity, specificity and precision but also development of suitable design of immunosensor for differential diagnosis of KB (T. indica). The approach involves the use of a mouse monoclonal antibody against diagnostic antigens and a suitable design of SPR sensor by the preparation of immuno-affinity layer over self assembled monolayer surfaces. The signal to noise ratio can further be enhanced using nano-gold particles. This will be first attempt for real-time monitoring of KB teliospores in wheat lots by SPR sensor and in a fully automated manner for establishing seed certification and plant quarantine standards.     

Multiplexed detection of antibodies to nonstructural proteins of foot-and-mouth disease virus

Liquid array technology was used to develop a multiplexed assay for the detection of antibodies to viral nonstructural proteins (NSPs), raised in cattle in response to infection with foot-and-mouth disease (FMD) virus. Two assays, one based on recombinant NSPs and the other on synthetically produced peptides, were developed and compared side-by-side. Serum samples from serial bleeds of cattle, each experimentally infected with one of the seven serotypes (C, A, O, Asia, SAT1, SAT2, SAT3) of FMD virus were analyzed. A distinct pattern in the detection of NSP antibodies and a close correlation of the recombinant protein and peptide-based assays were observed. The detection of antibodies to NSPs is a method to differentiate FMD-infected and FMD-vaccinated animals, and a high-throughput assay would be an invaluable tool in the case of an outbreak of FMD in North America, when emergency vaccination may be utilized to spare vaccinated, noninfected animals from slaughter and subsequent disposal.     

Fluoroimmunoassay for antigen based on fluorescence quenching signal of gold nanoparticles

A unique, sensitive, and highly specific fluoroimmunoassay system for antigen detection using gold and magnetic nanoparticles has been developed. The assay is based on the fluorescence quenching of fluorescein isothiocyanate caused by gold nanoparticles coated with monoclonal antibody. To demonstrate its analytical capabilities, the magnetic nanoparticles were coated with anti-alpha-fetoprotein polyclonal antibodies, which specifically bound with alpha-fetoprotein. Gold nanoparticles coated with anti-alpha-fetoprotein monoclonal antibodies could sandwich the alpha-fetoprotein captured by the magnetic nanoparticle probes. The sandwich-type immunocomplex was formed on the surface of magnetic nanoparticles and could be separated by a magnetic field. The supernatant liquid, which contained the unbound gold nanoparticle probes, was used to quench the fluorescence, and the fluorescence intensity of fluorescein isothiocyanate at 516 nm was proportional to the alpha-fetoprotein concentration. The result showed that the limit of detection of alpha-fetoprotein was 0.17 nM. This new system can be extended to detect target molecules with matched antibodies and has broad potential applications in immunoassay and disease diagnosis.     

Aptamer-based rolling circle amplification: a platform for electrochemical detection of protein

Aptamer-based rolling circle amplification (aptamer-RCA) was developed as a novel versatile electrochemical platform for ultrasensitive detection of protein. This method utilized antibodies immobilized on the electrode surface to capture the protein target, and the surface-captured protein was then sandwiched by an aptamer-primer complex. The aptamer-primer sequence mediated an in situ RCA reaction that generated hundreds of copies of a circular DNA template. Detection of the amplified copies via enzymatic silver deposition then allowed enormous sensitivity enhancement in the assay of target protein. This novel aptamer-primer design circumvented time-consuming preparation of the antibody-DNA conjugate for the common immuno-RCA assay. Moreover, the detection strategy based on enzymatic silver deposition enabled a highly efficient readout of the RCA product as compared to a redox-labeled probe based procedure that might exhibit low detection efficiency due to RCA product distance from the electrode. With the platelet-derived growth factor B-chain (PDGF-BB) as a model target, it was demonstrated that the presented method was highly sensitive and specific with a wide detection range of 4 orders of magnitude and a detection limit as low as 10 fM. Because of the wide availability of aptamers for numerous proteins, this platform holds great promise in ultrasensitive immunoassay.     

Sensitive and homogeneous protein detection based on target-triggered aptamer hairpin switch and nicking enzyme assisted fluorescence signal amplification

Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.     

Multiple fluorescence labeling with europium chelators. Application to time-resolved fluoroimmunoassays

Multiple fluorescence labeling with conventional probes like fluorescein, to improve the detection limit of labeled reactants, is not usually successful because of fluorescence quenching. In contrast, we found that the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) can be incorporated into proteins at very high molar ratios. Working with thyroglobulin as a model protein, we found that when 160 BCPDA molecules are incorporated into one thyroglobulin molecule, the fluorescence emitted by the labeled protein in the presence of excess Eu3+, is equivalent to that emitted by approximately 900 molecules of unconjugated BCPDA:Eu3+ complexes. We took advantage of the lack of any quenching effects and of the enhancement observed with the multiply labeled protein, to develop a universal reagent system consisting of (a) streptavidin covalently coupled to BCPDA labeled thyroglobulin and (b) excess Eu3+. With this approach, streptavidin is heavily labeled through thyroglobulin and retains its full biotin binding activity. We used the reagent to develop a highly sensitive time-resolved heterogeneous immunofluorometric assay of alpha-fetoprotein (AFP) in serum, using monoclonal antibodies. One antibody is immobilized in white microtitration wells (solid-phase) and the other is biotinylated. We demonstrate that this assay, using the newly developed reagent, is 25-fold more sensitive than the one using directly BCPDA labeled antibody and 5-fold more sensitive than an assay that uses BCPDA-labeled streptavidin. The detection limit of the assay with the new reagent was down to 60 amol of AFP per well. We conclude that multiple fluorescence labeling with europium chelators is an effective method of extending the sensitivity of currently used fluorescence immunoassay procedures.     

Dual detection of peptides in a fluorescence binding assay by employing genetically fused GFP and BFP mutants.

A competitive fluorescence microplate assay based on a red-shifted green fluorescent protein (rsGFP) and a blue fluorescent protein (BFP) was developed for the detection of two model peptides in the same sample. The assay employed gene fusion to prepare the fluorescently labeled peptide conjugates. Specifically, plasmids were constructed in which the genes encoding for the two small peptides (less than 12 amino acids in length) were fused to either the gene of the rsGFP or the BFP, as desired. The newly constructed plasmids were transformed into E. coli for expression of the fusion proteins. By employing the technique of gene fusion, one-to-one homogeneous populations of peptide-rsGFP or -BFP conjugates were produced. These peptide-GFP mutant conjugates exhibited the same excitation and emission spectral characteristics as the unmodified proteins. The naturally fluorescent proteins act as labels to provide sensitive dual detection of the two selected small peptides in a competitive assay format. To our knowledge, this is the first time that mutants of GFP, such as the rsGFP and BFP, have been used as quantitative labels for the development of a dual-analyte fluorescence immunoassay.     

Immunoassay readout method using extrinsic Raman labels adsorbed on immunogold colloids

An immunoassay readout method based on surface-enhanced Raman scattering (SERS) is described. The method exploits the SERS-derived signal from reporter molecules that are coimmobilized with biospecific species on gold colloids. This concept is demonstrated in a dualanalyte sandwich assay, in which two different antibodies covalently bound to a solid substrate specifically capture two different antigens from an aqueous sample. The captured antigens in turn bind selectively to their corresponding detection antibodies. The detection antibodies are conjugated with gold colloids that are labeled with different Raman reporter molecules, which serve as extrinsic labels for each type of antibody. The presence of a specific antigen is established by the characteristic SERS spectrum of the reporter molecule. A near-infrared diode laser was used to excite efficiently the SERS signal while minimizing fluorescence interference. We show that, by using different labels with little spectral overlap, two different antigenic species can be detected simultaneously. The potential of this concept to function as a readout strategy for multiple analytes is briefly discussed.     

Comparison of surface-enhanced resonance Raman scattering and fluorescence for detection of a labeled antibody

A comparison is made of the quantitative detection of a labeled antibody by surface-enhanced resonance Raman scattering (SERRS) and by fluorescence using the same instrument with the same laser excitation source. The area under the curve for the fluorescence band is greater than for any single peak in the SERRS spectrum, but the broad fluorescence band is more difficult to discriminate from the background at low concentrations. Using the peak height of one SERRS band and the peak height at the fluorescence maximum, the detection limit for SERRS was lower (1.19 x 10-11 mol.dm-3) than that obtained using fluorescence (3.46 x 10-10 mol.dm-3). The SERRS detection limit is calculated for the concentration of the sample added, but compared to fluorescence, there is an additional dilution step due to the addition of the colloid and the extent of this dilution is dependent on assay format. For comparison with the detection limits found earlier with labeled oligonucleotides, SERRS was remeasured with a 10 s accumulation time, and the final concentration in the cuvette after colloid addition and before any adsorption to the silver was used to calculate a detection limit of 2.79 x 10-13 mol.dm-3. This is comparable to the detection limit found using a similar SERRS procedure for an oligonucleotide labeled with the same dye. This experiment is dependent on many parameters that could affect this result, including the nature of the SERRS substrate, the excitation wavelength, and the dye chosen. However, the result indicates that SERRS can give assay sensitivities comparable or better than fluorescence for quantitative direct assay determination, suggesting that the much greater potential for multiple analyte detection could be exploited.     

Affinity assays using fluorescence anisotropy with capillary electrophoresis separation

A novel approach to detecting affinity interactions that combines fluorescence anisotropy with capillary electrophoresis (FACE) was developed. In the method, sample is injected into a capillary filled with buffer that contains a fluorescent probe that possesses low fluorescence anisotropy. If proteins or other large molecules in the sample bind the fluorescent probe, their migration through the capillary can be detected as a positive anisotropy shift. Thus, the method provides both separation and confirmation of binding to the probe. Calculations based on combining the Perrin equation and dissociation constant were used to predict the effect of conditions on aniostropy detection. These calculations predict that low probe concentrations yield the best sensitivity while higher concentrations increase the dynamic range for detection of binding partner. The assay was applied to detection of G proteins using BODIPY FL GTPgammaS as the fluorescent probe. Experimental measurements exhibited trends in anisotropy with varying probe and protein concentrations that were consistent with the calculations. The limit of detection for G(alphai1) was 3 nM when the electrophoresis buffer contained 250 nM BODIPY FL GTPgammaS. FACE affinity assay is envisioned as a method that can quantify selected binding partners and screen complex samples for compounds that possess affinity for a particular small molecule that is used as a probe.     

A Lipid-Nanopillar-Array-Based Immunosorbent Assay

Since infectious diseases, particularly viral infections, have threatened human health and caused huge economical losses globally, a rapid, sensitive, and selective virus detection platform is highly demanded. Enzyme-linked immunosorbent assay (ELISA) with flat solid substrates has been dominantly used in detecting whole viruses for its straightforwardness and simplicity in assay protocols, but it often suffers from limited sensitivity, poor quantification range, and a time-consuming assay procedure. Here, a lipid-nanopillar-array-based immunosorbent assay (LNAIA) is developed with a nanopillar-supported lipid bilayer substrate with fluorophore-modified antibodies for rapid, sensitive, and quantitative detection of viruses. 3D nanopillar array structures and fluid antibodies with fluorophores facilitate faster and efficient target binding and rapid fluorophore localization for quick, reliable analysis on binding events with a conventional fluorescence microscopy setup. LNAIA enables quantification of H1N1 virus that targets down to 150 virus particles with 5-orders-of-magnitude dynamic range within 25 min, which cannot be achieved with conventional ELISA platforms.     

Fluorescence anisotropy assay for the traceless kinetic analysis of protein digestion

A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of fluorescence anisotropy on the actual substrate of the protease. It takes advantage of epicocconone's ability to reversibly react with proteins to form an internal charge-transfer complex that is highly fluorescent. The protein-substrate is labeled in situ without the need for prior incubation and/or derivatization steps, which saves time and effort compared to methods employing specifically labeled protein-substrates. The assay can be carried out in 96- or 384-well plates, making it suitable for high-throughput applications in drug development and biotechnology.     

Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot‐Based Homogeneous FRET Immunoassay for Serum‐Based EGFR Detection

Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain thick polyethylene glycol (PEG)‐based coatings in order to retain the advantageous QD properties in biological media such as blood, serum or plasma. On the other hand, the application of QDs in Förster resonance energy transfer (FRET) immunoassays, one of the most sensitive and most common fluorescence‐based techniques for non‐competitive homogeneous biomarker diagnostics, is limited by such thick coatings due to the increased donor‐acceptor distance. In particular, the combination with large IgG antibodies usually leads to distances well beyond the common FRET range of approximately 1 to 10 nm. Herein, time‐gated detection of Tb‐to‐QD FRET for background suppression and an increased FRET range is combined with single domain antibodies (or nanobodies) for a reduced distance in order to realize highly sensitive QD‐based FRET immunoassays. The “(nano)²” immunoassay (combination of nanocrystals and nanobodies) is performed on a commercial clinical fluorescence plate reader and provides sub‐nanomolar (few ng/mL) detection limits of soluble epidermal growth factor receptor (EGFR) in 50 μL buffer or serum samples. Apart from the first demonstration of using nanobodies for FRET‐based immunoassays, the extremely low and clinically relevant detection limits of EGFR demonstrate the direct applicability of the (nano)^2‐ assay to fast and sensitive biomarker detection in clinical diagnostics.     

Glycerin suppression of fluorescence self-quenching and improvement of heterogeneous fluoroimmunoassay sensitivity

Fluorescent labels find wide application in immunoassays and immunosensors as well as in protein and DNA chips. However, the use of fluorescent labels in applications requiring high detection sensitivity is limited by fluorescence self-quenching observed when a relatively high number of fluorescent compounds is introduced in the recognition molecule. Here we describe a simple method that suppresses effectively fluorescence self-quenching observed when highly labeled antibodies are used as labels in immunoassays. This was achieved by treating the microtitration wells after the completion of the immunoassay with a glycerin solution followed by 15-min incubation of the emptied wells at 37 degrees C. The remedial action of this method on self-quenching was studied through a noncompetitive immunofluorometric assay for rabbit gamma-globulins employing a sheep anti-rabbit gamma-globulin antibody labeled with fluorescein at molar ratios ranging from 1.0 to 17.4. The glycerin/thermal treatment increased the fluorescence signal measured directly onto the solid surface by 9.2-117% for the antibodies with molar ratios of 1.0-17.4, compared with the values obtained prior to treatment. Furthermore, fluorescence self-quenching was completely removed for labeling ratios up to 14.0. The assay sensitivity was improved 2-4 times by the glycerin/thermal treatment when heavily fluoresceinated antibodies are used as labels (molar ratio >/=5.6). The proposed method resulted also in increased fluorescence signals when labels other than fluorescein were used and improved considerably the detection of protein spots on silicon dies.