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Metal-Enhanced Immunoassays

互联网2013-11-13

1329

实验原理

1. Metal-Enhanced Fluorescence

Discovery of surface-enhanced Raman scattering (SERS) (1 , 2 ) revitalized Raman spectroscopy and resulted in many new applications of the technology. In contrast to SERS, observed enhancement of fluorescence signal is not significant and often controversial due to strong quenching in a close proximity to the metal surface.

Metal-enhanced fluorescence (MEF) was demonstrated already three decades ago (3 –6 ). Recently, several groups reported fluorescence enhancements on various silvered surfaces, including silver island films (SIFs) (7 , 8 ), deposited colloids (9 , 10 ), photodeposited structures (11 ), and electron beam-deposited nanostructures (12 ). Although the enhancements were modest, usually in the order of tenfold as compared to many thousandfolds in SERS, the dependence of the enhancement on the distance from the metallic surface to fluorophores has been established (9 , 13 ). In general, the strongest enhancements are observed between 40 and 200 Å away from the metallic surface. At shorter distance the quenching dominates the fluorophore–metal interactions and at longer distances the enhancement gradually decreases. There are two enhancement effects: first is the enhanced local field, which is responsible for a higher excitation rate; and a second enhancement effect is due to an interaction of an excited molecule dipole with the nanoparticles, known as radiative decay engineering (RDE), which is responsible for a decrease of fluorescence lifetime. Total enhancement is the product of these two effects. The recent reviews (14 , 15 ) contain more details on MEF.

2. Model Immunoassays

To demonstrate a metal-enhanced fluorescence immunoassay on the glass surface coated with SIF, we used a model immunoassay format shown in Scheme 1. A model antigen, mouse Immunoglobulin G (IgG), was immobilized on the surfaces (SIF-coated surface and glass surface used as a reference), and corresponding specific antibodies labeled with a fluorophore were allowed to bind to the antigen. We incubated samples with antibodies for about 1 h to complete the binding reaction. Then the non-bound labeled antibodies were removed, a buffer was added, and fluorescence signal was measured in front-face configuration.

The measured fluorescence signal was a result of a specific interaction. To verify this, we performed the immunoassay using the same labeled antibodies, but with a nonspecific antigen, rabbit IgG, immobilized on the surface instead of the specific antigen. When a nonspecific antigen was used for the model immunoassay, no significant increase of the signal (non-specific binding) was observed. For more information about fluorescent immunoassays we refer reader to the recent review (16 ).

We found that silver colloids deposited on metallic mirrors show stronger enhancements than those deposited on glass substrates (17 , 18 ). It has been established that enhancements on sharp metallic edges are significantly more efficient (19 –21 ). Recently, we reported exceptionally strong local enhancements on self-assembled colloidal structures (SACS) formed on metallic films (22 ). This effect allows a significant reduction of the excitation intensity and effectively eliminates unwanted background from the regions away from the surface. The SACS looks similar to fractal-like structures with sharp edges. The interaction of local plasmons with metallic film results in strong local fields. A similar effect was recently described for a nanowire deposited on a metal film as an efficient platform for SERS (23 ).

In this chapter we will compare the fluorescence enhancements on SIFs and SACS. We will also emphasize the advantages of SACS in a model Alexa488 immunoassay. The immunoassays are ideal systems for observing strong fluorescence enhancements because the fluorescent probe is located at a close distance from the metallic surface.

实验试剂

1. PVA Films Doped with Fluorescein

Laser Grade Disodiumfluorescein was from Exction, Inc. Low molecular weight (13,000–23,000 MW) poly(vinyl alcohol) (PVA) was from Aldrich. Fluorescein-doped PVA samples were prepared on 1 × 1 in. microscope cover slips, either SIF coated or with SACS prepared on silver film.

2. Alexa488 Model Immunoassay

Mouse and rabbit IgGs, buffer components and salts (such as bovine serum albimun, sucrose) were from Sigma-Aldrich. Bocking solution was 1% bovine serum albumin, 1% sucrose, 0.05% NaN₃ , 0.05% Tween-20 in 50 mM Na-phosphate buffer, pH 7.3. Goat-anti-mouse antibodies labeled with AlexaFluor-488 were from Invitrogen, Inc. (dye/protein ratio 7). Microscope glass slides, 3 × 1 in., and 1 mm thick, were from VWR. Water was purified by Milli-Q system.

3. SIFs and SACS

Trisodium citrate was supplied by Spectrum. Silver nitrate and glucose were from Sigma-Aldrich. Microscope slides were coated by EMF Corp. (Ithaca, NY). A 52-nm thick layer of silver was deposited on the slide with about 2-nm chromium undercoat. The silver films were protected with 5 nm layer of silica. Poly-l-lysine solution was freshly prepared solution: 8 ml water 1.0 ml Na–phosphate buffer, 50 mM, pH 7.4, 1.0 ml poly-l-lysine solution (0.1%, Sigma).

实验步骤

1. Preparation of Fluorescein-Doped PVA Films

The 0.5% PVA solution containing disodiumfluorescein was spin-coated on slide substrates at 3,000 rpm. The thickness of the sample obtained with this preparation was about 20 nm (24 ).

2. Preparation of SIFs and SACS

1) SIFs Preparation

SIFs-coated surfaces were formed by an incomplete silver mirror reaction as described earlier (25 ). Glass slides were cleaned by soaking in H₂ SO₄ (98%) for 10–60 min and then were rinsed with Milli-Q water and dried on air before use. Slides were then coated with poly-l-lysine, with approximately 1.2 ml of the poly-l-lysine solution being added to each slide, incubated for 40–60 min and washed with water. Silver deposition was performed in a glass 100-ml beaker as follows. At intensive stirring, 20 drops of NaOH solution (5 M) were added to AgNO₃ solution (500 mg) in Milli-Q water (60 ml), and dark brown precipitate formed immediately. Approximately 2 ml of 30% NH₄ OH solution was added to dissolve the precipitate (at continuous intense stirring), and the clear solution was cooled with ice to 10–15°C (10 min). A fresh glucose solution (720 mg d( )glucose in 15 ml Milli-Q water) was added to the mixture, and glass slides were immediately half-inserted into the mixture. Soaking of slides was performed for pairs of slides, so only one surface of each slide was exposed to the reaction mixture. Stirring of the mixture was continued in an ice bath for 2 min, and then ice was removed and the solution was stirred at warming (i.e., at medium heating) until 30°C for approximately 2 min. Then the heating was turned off, and the solution continued to be mixed intensively for an additional 3–4 min (the temperature increased to 35°C). After the color of the slides became greenish-brown and the solution became opaque, the slides were removed from the beaker and washed with water two times with sonication for approximately 25 s.

2) SACS Preparation

Prior to SACS preparation, silver colloids were made as previously described (22 ). Briefly, all necessary glassware were soaked in a base bath overnight and washed scrupulously with deionized water. The solution of 0.18 mg/ml silver nitrate (200 ml) was heated and stirred in a 250 ml Erlenmeyer flask at 95°C. The first 0.5-ml aliquot of 34 mM trisodium citrate solution was added dropwise. The solution was stirred for 20 min and warmed to 96–98°C. Then five aliquots (0.7 ml each) of 34 mM trisodium citrate were added dropwise to the reaction mixture every 15–20 min. Stirring was continued for 25 min until the milky yellow color remained. Then the mixture was cooled in an ice bath for 15 min. After that silver colloids were used to prepare SACS as described earlier (22 ). Briefly, slide surfaces were cleaned and drop coated with silver colloids. The slides were air-dried to form SACS. The dry slides with self-assembled nanoparticles were stored and used within a month.

3. Alexa488 Model Immunoassay

Model immunoassay was performed on the slide surface as described earlier (25 ). Briefly, mouse IgG was non-covalently immobilized on the sample slide, or rabbit IgG on the control slide. Slides were covered with 20 μg/ml IgG solutions in 50 mM Na-phosphate buffer (pH 7.3) and incubated at room temperature overnight. Next, slides were rinsed with water and covered with blocking solution for 2 h at room temperature. Then, after rinsing with water, AlexaFluor-488 labeled anti-mouse antibody conjugate (33 nM in blocking solution) was added to the sample slides (with mouse IgG) or control slides (with rabbit IgG) and after 1 h incubation and washing, slides were covered with 50 mM Na-phosphate buffer (pH 7.3) and the fluorescence signal was measured.

4. Atomic Force Microscopy

Atomic Force Microscopy (AFM) was performed on an Explorer Scanning Probe Microscope (ThermoMicroscopes). Scanning was acquired in contact mode with Non-Conductive Silicone Nitride cantilever T: 0.59–0.61 μm (Veeco; MSCT-EXMT-A). Image data was processed and analyzed on Veeco DI SPMLab software.

The photographs, showing difference in intensity of Alexa488 signal on glass, SIF, and SACS, were taken with Canon 300D^® digital SLR camera. The illumination source was Ti: Sapphire laser (Mai Tai, Newport Corp, Irvine CA) coupled with SCG-800 photonic crystal fiber (Newport Corp, Irvine CA) for supercontinuum generation. 470-nm band pass filter was used to select appropriate wavelength from the supercontinuum and the photographs were taken through 495 long-pass filter. For all the photographs, exposure time of one-fourth of a second and aperture of “14” was used. All images were cropped equally to one-fourth of the original image using ZoomBrowser^® software (Canon Inc.).

6. Fluorescence Measurements

Fluorescence spectra and lifetimes were measured on FluoTime200 fluorometer (PicoQuant, GmbH). This instrument is equipped with a monochromator and a microchannel plate photomultiplier (MCP) detector on the observation path. With a 470-nm laser pulsed laser diode (pulse duration 68 ps) excitation, this fluorometer is capable of resolving sub-nanosecond intensity decays.

7. Microscopy Measurements

The Time-Correlated Single Photon Counting (TCSPC) MicroTime 200 confocal system (PicoQuant, GmbH., Berlin, Germany) coupled to Olympus IX71 microscope was used for measuring fluorescence enhancements. The fluorescence was excited with pulses from picosecond laser diode 470 nm (LDH-P-C-470B) worked at 20 MHz repetition rate and detected by Photon Avalanche Diode (SPAD) Perkin-Elmer (SPCM-AQR-14) detector. The fluorescence was observed confocally from the sample spin-coated on cover slips. Special, non-fluorescing cover slips were used (Menzel-Glasser #1). Sample was placed on the microscope stage, and light was focused (Olympus water immersion objective NA1.2, 60× magnification) in the sample plane. The observation path was equipped with three long-pass filters 500 nm to block excitation light and count fluorescence of the dye only. Photons were counted by TCSPC PicoHarp 300 board. The data was stored in the time-tagged time-resolved mode, which allowed the recording of every detected photon with its individual timing and detection channel information. All measurements and data analysis were performed using SymPho Time Software v. 5.0.

8. Interpretation of Measurements

1) Fluorescein-Doped PVA Films

The magnitude of a fluorescence enhancement depends on the distance of fluorophores from the metallic surface as well as on a quantum yield of the probe. The strongest dependence, however, is on the metallic surface structure. The most popular and easy for the preparation are SIFs. Figure 1 shows typical SIFs deposited on a glass substrate evaluated by AFM. The surface is relatively homogeneous. The SACS prepared on a silver film show a different surface morphology (Fig. 2). The silver elongated nanoparticles form fractal-like structures (Fig. 2, left), making the metallic surface very heterogeneous. The expectation is to observe on these surfaces non-uniform enhancements, in contrast to glass and SIFs surfaces.

Emission spectra of fluorescein in PVA spin-coated on glass, SIFs, and SACS substrates are shown in Fig. 3. The spectrum on SACS is not only the strongest but also shifted towards longer wavelengths. Such shift is expected in very strong fields (26 ) and has been already observed (22 ). The comparison of the brightness between samples on glass, SIFs, and SACS are shown in the Fig. 4. The photographs were taken at the same excitation and observation conditions.

Next, we collected confocal images from these samples (Fig. 4, top panels).

The fluorescence intensities on SIF and SACS substrates are much stronger than on the glass. Also, the fluorescence on SACS is localized in “hot spots” where the enhancement is higher. Figures 5 and 6 show the intensity and lifetime traces across the images as indicated by the lines in Fig. 4, top panels. As expected, the distributions of intensities and lifetimes on glass and SIFs substrates are relatively homogeneous. In contrast, on the SACS substrate, both intensity and lifetime traces are heterogeneous. Also, as expected, the stronger brightness corresponds to the shorter lifetime, see arrows on Fig. 6. This indicates that RDE effect plays an important role in the total enhancement. The lifetime images and distributions from entire 30 × 30 μm² areas are shown in Fig. 7. This figure visualizes changes in the lifetime induced by metallic nanoparticles. More precise lifetime measurements are shown on Fig. 8. The amplitude averaged lifetimes are significantly shorter on metallic surfaces than on the glass.

2) Alexa488 Model Immunoassay

We performed a model immunoassay on glass and SACS substrates using an Alexa488 fluorescent probe. Emission spectra (Fig. 9) show more than tenfold enhancement on SACS compared to the glass. The fluorescence signal from a control (non-specific binding) was about 10% of the sample signal in both glass and SACS substrates. The distributions of intensities and lifetimes on SACS substrate are shown on Fig. 10. Again, higher intensity corresponds to shorter lifetime, and in the less bright spots lifetimes are longer. Although the average enhancement is not high (see Fig. 9), in the local “hot spots” (Fig. 10, top), the brightness is about 100-fold higher than in the absence of silver a nanostructure.

The Alexa488 lifetime distributions on glass and SACS are shown in Fig. 11. The average lifetime on SACS is a few times shorter than on glass. This observation was confirmed in lifetime measurements with a high-resolution fluorometer (not shown). Finally, we compared the photostabilities of Alexa488 immunoassays on glass and SACS (Fig. 12). The shorter lifetimes are responsible for higher photostabilities on metallic surfaces. The photodegradation occurs mostly in the excited state, and near metallic nanostructure molecules can emit many photons before they are bleached.

注意事项

The surface preparation is crucial in MEF and has to be done very carefully.

For detection purposes in macroscopic devices SIFs surfaces perform well, providing a uniform enhancement in large areas. In the case of microscopy measurements, like in single molecule detection (SMD), the SACS substrates are more useful because they offer “hot spots” with unprecedented fluorescence enhancements. Observation of “hot spots” enables a significant reduction of the excitation power. In turn, this reduces background which often makes SMD measurements difficult or impossible.

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