DESC:AN APPARTUS FOR ENHANCED FLOURESCENCE DETECTION

FIELD OF INVENTION
The invention generally relates to the field of sensing and imaging and particularly to an apparatus for enhanced fluorescence detection.

BACKGROUND
Detecting weak signals have always been a challenge in the field of sensing and imaging specifically so, in the detection of fluorescence signals. Several substrates are available for enhancing a fluorescing signal. One such substrate includes based on 3D network forming porous layers which immobilize the receptor molecules required for capture of analyte molecules from sample. Due to their porosity and enhanced surface roughness, they capture significantly more receptor molecules on the sensor surface. However, these 3D porous functionalization layers are proprietary coatings which are expensive and more importantly suitable for only one type of surface, i.e. either glass or gold etc. The other commercially available technology for fluorescence enhancement is based on multilayer dielectric coatings deposited on glass substrates such that an electric field anti-node condition is established at the top surface of the multilayer stack. In this approach typically about 20 quarter-wave thick layers of alternating high and low refractive indices are coated on a substrate using expensive vacuum coating tools with precisely controllable deposition parameters. The disadvantages of this approach are the cost of installing vacuum deposition systems and the inability to have good quality coatings on plastic surfaces. The cost per sensor is quite high due to the manufacturing process aforementioned and may not be suitable to develop low cost disposable diagnostic chips.

BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG.1 illustrates an apparatus for enhanced fluorescence detection according to an embodiment of the invention.
FIG. 2 shows a comparison of fluorescence intensities of a sample as measured through existing substrate and through the apparatus, according to an embodiment of the invention.

SUMMARY OF THE INVENTION
One aspect of the invention provides a method for enhanced fluorescence detection. The method includes selecting a substrate. The selected substrate is then layered with a first polyelectrolyte having a first polarity. A second layer is deposited on top of the first layer with a second polyelectrolyte having a second polarity. Each of the first polyelectrolyte and the second polyelectrolyte is alternatively layered to obtain a multiple polyelectrolyte layer. The effective thickness of the multiple polyelectrolyte layer is quarter of a wavelength. The wavelength selected for determining the thickness of the polyelectrolyte layer is that of incident electromagnetic radiation. The multiple polyelectrolyte layer having alternating polarities enhances the fluorescence.
Another aspect of the invention provides a system for enhanced fluorescence detection. The system includes a substrate, a plurality of polyelectrolyte layered on the substrate, a light source for illuminating the polyelectrolyte layers and a means for detecting the emitted fluorescence signals.

DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a method and a system for enhanced fluorescence detection. The method includes selecting a substrate, then layering the substrate with at least two polyelectrolyte layers having alternating polarity, illuminating the polyelectrolyte layers with a light source and detecting the emitted fluorescence signals. The apparatus includes a fluorescence enhancement substrate coated with a quarter-wave thick polyelectrolyte layer to create the electric field anti-nodal surface enhancing the fluorescence signal. The polyelectrolyte layers, being porous, increases the fluorescence signal due to increased surface area. The increased surface area enhances mass loading as well as 3D capture across the layer thickness. The construction of the apparatus along with the method of use of the apparatus shall be described in detail herein below.
FIG.1 illustrates an apparatus for enhanced fluorescence detection according to an embodiment of the invention.
The apparatus includes a substrate 101 and a plurality of polyelectrolyte 103 layered on the substrate. The substrate 101 is a metallic surface. Alternatively, the substrate is a non-metallic surface coated with a thin layer of a metal. The example of non-metallic surface includes but is not limited to glass, plastics such as polycarbonate and polystyrene. In one embodiment of the invention, the substrate 101 is obtained by coating a metallic layer on a non metallic surface. An example of such a substrate includes but is not limited to a metal coated glass slide and a metal surface such as an aluminum foil.
The substrate 101 is then coated with a plurality of polyelectrolyte layers 103. Each of the layers of the polyelectrolyte comprises of charged polymers in aqueous solution. In one embodiment of the invention, each of the layers of polyelectrolyte is alternately charged. Further, each of the layers of polyelectrolyte is deposited on the substrate by dipping the substrate into a solution of the polyelectrolyte for a predefined duration. Alternatively, the deposition of the layer of polyelectrolyte is achieved through spin coating, spray coating and all such processes known to a person skilled in the art. In addition all these techniques are also compatible with photolithographic patterning. The same techniques can also be used to deposit polyelectrolyte on flexible substrates such as aluminum foils. Examples of polyelectrolyte include but are not limited to Polyacrylic acid (PAA), Polyallylamine hydrochloride (PAH), Polystyrene sulfonate (PSS), Polydiallyl dimethyl ammonium chloride (PDDAC), Polyethyleneimine (PEI). The process of depositing alternatively charged layers of polyelectrolytes creates an anti-nodal condition results in enhancing the fluorescence excitation and emission rates causing increased fluorescence signal. The total thickness of the plurality of layers of the polyelectrolyte is equal to a quarter of a wavelength used for fluorescent excitation.
The invention also provides a method for enhanced florescence detection. The method includes selecting a substrate, layering the substrate with at least two polyelectrolyte layers having alternating polarity, illuminating the polyelectrolyte layers with a light source and detecting the emitted fluorescence signals. Light of desired wavelength falls on the substrate 101 layered with a plurality of polyelectrolyte layers 103. The polyelectrolyte layers, being porous, increases the fluorescence signal due to increased surface area. The increased fluorescence signals are then detected by a detector. The increased surface area of the polyelectrolyte layer enhances mass loading as well as 3D capture across the layer thickness. Further, the advantage of using a polyelectrolyte layer is that proteins can diffuse through the entire thickness of the film as shown in Fig. 1 (b). Therefore, in addition to the electric field antinodal condition obtained with the quarter-wavelength thickness of the polyelectrolyte layers, a higher loading of the surface is also obtained due to this depth wise diffusion.
Fig. 2 shows comparison of fluorescence intensities of a sample as measured through existing substrates and through the apparatus, according to an embodiment of the invention.
The data points marked Glass-(PAH/PAA) 10.5 in Fig. 2 (a) denotes the fluorescence signal obtained by coating a quarter-wavelength thick polyelectrolyte layers on glass which does not support fluorescence enhancement theoretically. However, when the same thickness polyelectrolyte is deposited by any of the techniques described above, the fluorescence signal increases confirming the theoretically expected fluorescence enhancement. This data point is denoted as Al-(PAH/PAA) 10.5 in the figure. A commercially available substrate for enhanced fluorescence is also shown by way of comparison. The metal coated polyelectrolyte layers (as provided by the invention) provide similar performance to the commercially available product which is denoted as FAST slide in the picture.
Figure 2 (b) is same as Fig. 2 (a) except the data is referenced to a baseline, which is taken as a glass slide with a single polyelectrolyte layer in which case there is no fluorescence enhancement either from metal or from the depth wise diffusion process shown in Fig. 1 (b). Figures 2 (c) to (f) corresponds to the actual images obtained from the microscopes corresponding to the data points in Fig. 2 (a). The four different pictures correspond to the four data types referred in Fig. 2 (a) in the same order.
The technique described here for fluorescence enhancement can be used broadly in the area of fluorescence based diagnostics, for e.g. detection of biomarkers as indicators of diseased state, pathogens such as bacteria and other microorganisms, DNA and protein microarrays.
The invention also provides a method for obtaining any substrate as the attachment of the polyelectrolyte functionalization layer is based on surface charge and not on the surface chemical properties. As a result the method here can be applied to a variety of substrates ranging from glass, any metal surface and quite importantly, low cost plastics such as polycarbonate or polystyrene.
The invention thus provides for an apparatus for enhanced fluorescence which is easy to manufacture and cost effective. Moreover, the apparatus provided by the invention can be configured to form a flexible substrate and is capable of being formed as continuous roll in a roll-to-roll manufacturing process.
The foregoing description of the invention has been set for merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.
,CLAIMS:We Claim:
1. A method for obtaining enhanced fluorescence, the method comprising
selecting a substrate;
layering the substrate with a first polyelectrolyte layer having a first polarity;
layering the first polyelectrolyte layer with a second polyelectrolyte layer having a second polarity;
alternatively layering the first polyelectrolyte layer and the second polyelectrolyte layer until a multiple polyelectrolyte layer is obtained; and
detecting the emitted fluorescence signals from the multiple polyelectrolyte layer.
2. The method of claim 1, wherein the first polarity of the first polyelectrolyte layer is opposite to the second polarity of the second polyelectrolyte layer.
3. The method of claim 1, wherein the multiple layer of the polyelectrolyte has an effective thickness of about quarter of a wavelength.
4. The method of claim 1, wherein the wavelength selected for determining the thickness of the polyelectrolyte layer is that of incident electromagnetic radiation.
5. The method of claim 1, wherein the substrate is either a metal based substrate or a non-metal based substrate coated with a metal.
6. The method of claim 1, wherein each of the polyelectrolyte layer is selected from a group comprising of a polyacrylic acid, a polyallylamine hydrochloride, a polystyrene sulfonate, a polydiallyl dimethyl ammonium chloride and a polyethyleneimine .
7. The method of claim 1, wherein the step of layering the polyelectrolyte is achieved by at least one technique selected from the list comprising of dipping, spray coating, spin coating and photolithographic patterning.
8. A system for enhanced fluorescence detection, the system comprising
a substrate;
a multiple polyelectrolyte layer formed on the substrate;
an electromagnetic radiation source for illuminating the polyelectrolyte layer; and
a detector for detecting emitted fluorescence signals.
9. The system of claim 8, wherein the substrate is a metallic reflecting substrate.
10. The system of claim 8, wherein the multiple polyelectrolyte layers comprises of at least two polyelectrolytes with opposing polarity.